Bzip type transcription factors regulating the expression of rice storage protein

ABSTRACT

cDNAs (RISBZ1, RISBZ4, and RISBZ5) encoding bZIP transcription factors were isolated from a cDNA library originating in rice plant seed. The cDNAs encode novel proteins and have binding activity to the GCN4 motif. Among them, RISBZ1 activated transcription mediated by the GCN4 motif by 100-fold or more. Since the expression of RISBZ1 precedes the expression of a seed storage protein gene and is expressed only in maturing seeds, it is suggested that RISBZ1 controls the expression of rice seed storage proteins. In addition, by linking the recognition sequence of the transcription factor, the GCN4 motif, in tandem and introducing it into the promoter for a gene encoding seed storage protein to facilitate its binding to the transcription factor RISBZ1, expression of a foreign gene under the control of the modified promoters is greatly enhanced.

TECHNICAL FIELD

[0001] The present invention relates to a novel transcription factor andits use pertaining to the endosperm-specific expression of the storageprotein in the rice plant seed.

BACKGROUND ART

[0002] Seed storage protein is expressed in seeds only during thematuring stage, and the expression of genes encoding this protein isanalyzed as a suitable model for investigating the transcriptionregulatory mechanism of plant genes (Goldberg, R. B. et al., Science266: 605-614, 1994). The expression of a gene that codes for a seedstorage protein is known to be regulated by the cooperation of aplurality of cis factors in a promoter. The binding of a transcriptionfactor to a specific cis regulatory factor is important in theinitiation of transcription and the tissue- and time-specificexpression. It can be explained that the expression of a seed storageprotein is induced by several types of cis regulatory factors relatingto the regulation of seed-specific expression when transcription factorsthat recognize specific cis regulatory factor bind and aggregate.Functional analyses of cis regulatory factors and transcription factorsof crop storage protein genes have been conducted in order to elucidatethe molecular mechanism of the expression of seed storage proteins(Thomas, T. L., Plant Cell 5: 1401-1410, 1993; Morton, R. L. et al., inSeed Development and Germination, pp. 103-138, Marcel Dekker, Inc.,1995).

[0003] However, despite considerable research, analyses usingtransformed plants failed to identify the cis regulatory factorsessential for gene expression regulation in nearly all crops studied,and the gene expression regulatory mechanism has still not been clearlyunderstood. In the case of monocotyledons in particular, the promoteranalyses using stable transformed plants has been performed in only theseed storage protein, glutelin, of the rice plants. On the other hand,in the case of maize, wheat and barley, analyses have been conductedusing particle guns or tobacco transformants (Muller, M. and Knudsen,S., Plant J. 6:343-355, 1993; Albani, D. et al., Plant Cell 9: 171-184,1997; Marzabal, P. M. et al., Plant J. 16: 41-52, 1998).

[0004] It has been shown that the endosperm-specific expression of theseed storage protein gene of grains is controlled by the collaborativeaction of several types of cis regulatory factors. The Prolamin box(TGTAAAG), GCN4 motif (TGA(G/C)TCA), AACA motif (AACAAAA), and ACGTmotif, which are conserved in the seed storage protein gene promoters ofnumerous grains, have been characterized as cis regulatory factorsinvolved in endosperm-specific expression by loss-of-function andgain-of-function analyses (Morton, R. L. et al., In: Seed Developmentand Germination, pp. 103-138, Marcel Dekker Inc., 1995).

[0005] The GCN4 motif has been frequently found not only from seedstorage protein gene, but also from promoters of genes involved in themetabolism (Muller, M. and Knudsen, S., Plant J. 6: 343-355, 1993).Recently, a polymer of the GCN4 motif of rice plant glutelin gene hasbeen found to reproduce endosperm-specific expression in transformedrice plants, and remarkable decrease in promoter activity and changes inits expression pattern have been found due to the substitution ordeletion of nucleotides in the GCN4 motif. These facts prove that theGCN4 motif plays an important role in endosperm-specific expression (Wu,C. Y. et al., Plant J. 14: 673-683, 1998). The GCN4 motif is coupled toa Prolamin box (TGTAAAG) via a plurality of bases in many cases, and isone of the constituents of the two-factor endosperm box found in theprolamin gene promoters of nearly all grains, including wheat glutenin,barley hordein, rye secalin, sorghum cafulin and adlay coixin. The AACAmotif is involved in the expression of nearly all rice glutelin genes.Although the combination of two motifs (GCN4 motif and Prolamin box orGCN4 motif and AACA motif) is required for gene expression, in order toadequately function as an endosperm-specific promoter, an additionalmotif is essential (Takaiwa, F. et al., Plant Mol. Biol. 30: 1207-1221,1996; Yoshihara, T. et al., FEBS Letts. 383: 213-218, 1996; Wu, C. Y. etal., Plant J. (in press)). Recently, it has been demonstrated that, inorder to function as aminimum promoter capable of reproducingendosperm-specific expression in glutelin genes (GluB1) of rice plant,at least three constituents, the GCN4 motif, the AACA motif, and theACGT motif, present in the −197 bp promoter region, are essential (Wu,C. Y. et al., Plant J. 14: 673-683, 1998; Wu, C. Y. et al., Plant J. 23:415-421, 2000).

[0006] Opaque2 (O2) of maize is an endosperm-specific transcriptionfactor of the bZIP type, and this O2 binds to the ACGT motif in the 22kDa α-zein gene promoter of maize to activate transcription (Schmidt, R.J. et al., Plant Cell 4: 689-700, 1992). O2 has been reported to beinvolved in endosperm-specific transcription of b-32 ribosomedeactivating protein gene by binding to the (Ga/tTGAPyPuTGPu) sequence(Lohmer, S. et al., EMBO J. 10: 617-624, 1991). O2 is thus considered tohave a wide-ranging binding capability. Reportedly, the GCN4 motif isrecognized by O2, and transcription is activated through the binding ofO2 to the GCN4 motif (Wu, C. Y. et al., Plant J. 14: 673-683, 1998;Holdsworth, M. J. et al., Plant Mol. Biol. 29: 711-720, 1995). In seeds,during the maturing stage, in vivo footprint analysis showed that thenuclear protein binds to the GCN4 motif and Prolamin box present inwheat low molecular weight glutenin gene promoter (Vicente-Carbajos, J.et al., Plant J. 13: 629-640, 1998) and maize γ-zein gene promoter(Marzabal, P. M. et al., Plant J. 16: 41-52, 1998). In addition, theresults of an in vitro DNaseI footprint analysis showed that the nuclearprotein of maturing rice plant seeds as well as GST-02 fused proteinspecifically recognize the GCN4 motif of the rice glutelin gene promoter(Wu, C. Y., et al., Plant J. 14: 673-683, 1998; Kim, S. Y. and Wu, R.,Nucl. Acids Res. 18: 6845-6852, 1990). These findings indicate that anO2-like transcription factor is present in grain seeds, and that itcontrols the endosperm-specific expression of numerous seed storageprotein genes mediated by the GCN4 motif.

[0007] Recently, cDNA clones of transcription factors that recognize theGCN4 motif have been isolated in wheat (Albani, D. et al., Plant Cell9:171-184, 1997) and barley (Vicente-Carbajos, J. et al., Plant J. 13:629-640, 1998; Onate, L. et al., J. Biol. Chem. 274:9175-9182, 1999),and have been named SPA, BLZ1 and BLZ2. These transcription factors havebeen determined to activate the transcription of seed storage proteingenes mediated by the GCN4 motif in wheat low molecular weight gluteninand barley B1 hordein gene promoter. Interestingly, these transcriptionfactors were expressed seed-specifically. Although cDNA that codes for atranscription factor having a high homology with the bZIP domain of O2has previously been isolated from rice plants, it remains to beconfirmed whether or not it activates transcription of seed storageprotein gene mediated by the GCN4 motif (Izawa, T. et al., Plant Cell 6:1277-1287, 1994; Nakase, M. et al., Plant Mol. Biol. 33: 513-522, 1997).

DISCLOSURE OF THE INVENTION

[0008] An object of the present invention is to provide a noveltranscription factor that regulates the expression of rice seed storageprotein by binding to the GCN4 motif, a gene that codes for the factor,plant cells and plant bodies in which the gene has been introduced, anda method for production and use thereof.

[0009] The present inventors conducted research to resolve the aboveproblems. As mentioned above, the GCN4 motif is a sequence that ishighly conserved in the promoters of grain seed storage protein genes,and plays a central role in the endosperm-specific expression of thegenes. This GCN4 motif is recognized by the bZIP transcription factorfamily that is closely related to the Opaque2 (O2) protein of maize.Therefore, the present inventors thought that, by isolating bZIPtranscription factor from the rice seeds, it would be possible toidentify the transcription factor that binds to the GCN4 motif tocontrol the expression of rice seed storage protein.

[0010] First, the present inventors screened a cDNA library originatingin rice seed and isolated cDNA that codes for five types of bZIPtranscription factors (RISBZ1, RISBZ2, RISBZ3, RISBZ4, and RISBZ5).Based on the homology of the presumed amino acid sequences, RISBZ2 andRISBZ3 were identical to RITAL (Izawa, T. et al., Plant Cell 6:1277-1287, 1994) and REB (Nakase, M. et al., Plant Mol. Biol. 33:513-522, 1997), respectively, and the remaining RISBZ1, RISBZ4; andRISBZ5 were revealed to code for novel proteins. When the bindingability of RISBZ1, RISBZ2, RISBZ3, RISBZ4, and RISBZ5 to GCN4 motif wasinvestigated, they all exhibited binding activity to the GCN4 motif.Furthermore, the transcription activation ability of the five proteinsby binding to the GCN4 motif was investigated. As a result, only RISBZ1activated transcription 100-fold or more by binding to the GCN4 motif.In addition, an analysis using the GAL4 DNA binding domain of yeastrevealed that proline-rich, 27 amino acid residues of the N-terminalside of RISBZ1 functioned as a the transcription-activating domain. Thedifference in transcription activation ability between RISBZ1 and theother RISBZ proteins was primarily due to the mutation of 7 amino acidresidues (for RISBZ2) or deletion of the transcription-activating domain(for RISBZ3, RISBZ4, and RISBZ5). This finding suggests that thedifference in transcription activation ability between RISBZ1 and otherRISBZ proteins occur due to a structural mutation of the transcriptionactivating domain. In addition, RISBZ1 was found to form not only ahomodimer, but also heterodimers with other RISBZ proteins. Since theexpression of RISBZ1 precedes the expression of seed storage proteingene and is expressed only in maturing seeds, RISBZ1 may control theexpression of seed storage protein. In order to investigate theexpression of RISBZ1 gene, the promoter of the RISBZ1 gene was coupledto a GUS reporter gene, and this construct was introduced into a riceplant. In this rice plant the GUS gene was strongly expressed in thealeurone layer.

[0011] As described above, the present inventors demonstrated that thenovel proteins RISBZ1, RUSBZ4, and RISBZ5 actually bind to the GCN4motif, and clarified that RISBZ1 is a transcription activation factorinvolved in endosperm-specific expression of the rice seed storageprotein gene.

[0012] The present inventors also produced a transformed plant thatcontained a DNA construct in which the RISBZ1 of the present inventionwas connected downstream of a promoter and a DNA construct in which areporter gene was connected downstream of a promoter containing thetarget sequence of RISBZ1. The inventors then succeeded in measuring thetranscription activity of RISBZ1 in the transformed plant by using theexpression of the reporter gene as an indicator. These findings enablehigh level expression of a useful, highly value-added foreign genewithin the transformed plant cells in which the foreign gene isconnected downstream of a promoter containing the target sequence ofRISBZ1 instead of the above reporter gene.

[0013] The present invention relates to a novel transcription factorthat regulates the expression of rice seed storage protein by binding tothe GCN4 motif, a gene encoding the factor, plant cells and plants inwhich the gene has been introduced, and methods for production and usethereof. More specifically, the present invention provides thefollowing:

[0014] [1] a DNA selected from the following (a) through (d):

[0015] (a) a DNA encoding a protein comprising the amino acid sequenceset forth in any one of SEQ ID NOs: 2, 5, and 7;

[0016] (b) a DNA comprising a coding region of the nucleotide sequenceset forth in any one of SEQ ID NOs: 1, 3, 4, and 6;

[0017] (c) a DNA comprising the amino acid sequence set forth in any oneof SEQ ID NOs: 2, 5, and 7, in which one or more amino acids aresubstituted, deleted, added, and/or inserted, and encoding a proteinthat is functionally equivalent to a protein comprising the amino acidsequence set forth in any one of SEQ ID NOs: 2, 5, and 7; and

[0018] (d) a DNA hybridizing under stringent conditions with a DNAcomprising the nucleotide sequence set forth in any one of SEQ ID NOs:1, 3, 4, and 6, and encoding a protein functionally equivalent to aprotein comprising the amino acid sequence set forth in any one of SEQID NOs: 2, 5, and 7;

[0019] [2] the DNA according to [1], which encodes a protein that bindsto the GCN4 motif or activates expression of rice seed storage protein;

[0020] [3] the DNA according to [1] or [2], which is derived from riceplant;

[0021] [4] a DNA encoding antisense RNA complementary to a transcriptionproduct of the DNA according to any one of [1] through [3];

[0022] [5] a DNA encoding an RNA having ribozyme activity thatspecifically cleaves a transcription product of the DNA according to anyone of [1] through [3];

[0023] [6] a DNA encoding an RNA that suppresses the expression of theDNA according to any one of [1] through [3] in plant cells byco-inhibition effects, and having 90% or more homology with the DNAaccording to any one of [1] through [3];

[0024] [7] a DNA encoding a protein having a dominant negative phenotypeof a protein encoded by the DNA according to any one of [1] through [3]which is endogenous in plant cells;

[0025] [8] a vector containing the DNA according to any one of [1]through [3];

[0026] [9] a transformed cell retaining the DNA according to any one of[1] through [3] or the vector according to [8];

[0027] [10] a protein that is encoded by the DNA according to any one of[1] through [3];

[0028] [11] a method of producing the protein according to [10], themethod comprising steps of culturing the transformed cell according to[9] and collecting the expressed protein from said transformed cell ortheir culture supernatant;

[0029] [12] a vector containing the DNA according to any one of [4]through [7];

[0030] [13] a transformed plant cell retaining the DNA according to anyone of [1] through [7] or the vector according to [8] or [12];

[0031] [14] a transformed plant containing the transformed plant cellaccording to [13];

[0032] [15] a transformed plant that is a progeny or clone of thetransformed plant according to [14];

[0033] [16] a reproductive material of the transformed plant accordingto [14] or [15];

[0034] [17] an antibody that binds to the protein according to [10];

[0035] [18] a plant having on its genome a DNA construct in which theDNA according to [1] is operably connected downstream of an expressioncontrol region and a DNA construct in which a foreign gene is operablyconnected downstream of an expression control region having the targetsequence of the protein according to [10];

[0036] [19] the plant according to [18], wherein the target sequence isa sequence containing the GCN4 motif;

[0037] [20] the plant according to [19], wherein the GCN4 motif has thesequence set forth in any one of SEQ ID NOs: 8, 13, and 14;

[0038] [21] the plant according to [18], wherein the target sequence isa sequence containing a G/C box; and,

[0039] [22] a method of producing the plant according to any one of [18]through [21], the method comprising a step of crossing a plant having onits genome a DNA construct in which the DNA according to [1] is operablyconnected downstream of an expression control region, with a planthaving on its genome a DNA construct in which a foreign gene is operablyconnected downstream of an expression control region containing thetarget sequence of the protein according to [10].

[0040] The present invention provides DNAs encoding RISBZ1, RISBZ4, andRISBZ5 protein originating in the rice plant. The nucleotide sequence ofthe cDNA of RISBZ1 is shown in SEQ ID NO: 1, the amino acid sequence ofthe protein encoded by the cDNA is shown in SEQ ID NO: 2, and thenucleotide sequence of the genome DNA is shown in SEQ ID NO: 3 (thegenome DNA sequence set forth in SEQ ID NO: 3 contains introns and iscomposed of six exons). The nucleotide sequences of the cDNAs of RISBZ4and RISBZ5 proteins are shown in SEQ ID NO: 4 and 6, respectively, whilethe amino acid sequences of the proteins encoded by the cDNAs of RISBZ4and RISBZ5 proteins are shown in SEQ ID NO: 5 and 7, respectively. Inthe present specification, the RISBZ1, RISBZ4, and RISBZ5 of the presentinvention are collectively referred to as RISBZ.

[0041] The RISBZ proteins of the present invention are thought to bebZIP transcription factors having the ability to bind the GCN4 motif.Among these, RISBZ1 remarkably activates transcription by binding to theGCN4 motif. Since the promoter of the RISBZ1 gene is activated in thealeurone layer of rice seeds, RISBZ1 is thought to be atranscription-activating factor that controls the expression of riceseed storage protein.

[0042] In addition, it has been reported that bZIP transcription factorsform various homo/heterodimers through the combination of variousfactors belonging to the bZIP transcription factor family. As a result,control factors with various functions are formed, which control genetranscription. In the Examples described below, RISBZ2 and RISBZ3 wereshown to form a heterodimer with RISBZ1. In addition, RISBZ4 and RISBZ5have extremely high homology (96% and 82.7%, respectively) with the bZIPdomain of RISBZ3, and these factors would also form heterodimers withRISBZ1. These facts suggest that RISBZ4 and RISBZ5 of the presentinvention would form, with the RISBZ1 and other RISBZ members of thepresent invention, heterodimers having various transcription activatingabilities and DNA binding properties depending on the maturation stageand tissue to control the expression of seed storage protein.

[0043] Thus, the DNA encoding the RISBZ protein of the presentinvention, or a molecule that controls the expression of the DNA, wouldbe useful in, for example, regulating the expression of seed storageprotein. Regulation of the expression of seed storage protein hasvarious industrial advantages. For example, it would be possible toaccumulate abundant foreign gene products in the endosperm by deletingseed storage protein in the endosperm. On the other hand, by highlyaccumulating seed storage protein in the endosperm, it would be possibleto produce seeds (e.g., rice) having greater nutritional value.

[0044] The DNA encoding the RISBZ protein of the present inventionincludes genomic DNA, cDNA, and chemically synthesized DNA. A genomicDNA and cDNA can be prepared according to conventional methods known tothose skilled in the art. More specifically, a genomic DNA can beprepared, for example, as follows: (1) extracting genomic DNA from plantcells or tissues; (2) constructing a genomic library (utilizing avector, such as plasmid, phage, cosmid, BAC, PAC, and so on); (3)spreading the library; and (4) conducting colony hybridization or plaquehybridization using a probe prepared based on the DNA encoding theprotein of the present invention (e.g. SEQ ID NO: 1, 3, 4, or 6).Alternatively, a genomic DNA can be prepared by PCR, using primersspecific to the DNA encoding the protein of the present invention (e.g.SEQ ID NO: 1, 3, 4, or 6). On the other hand, cDNA can be prepared, forexample, as follows: (1) synthesizing cDNAs based on mRNAs extractedfrom plant cells or tissues; (2) preparing a cDNA library by insertingthe synthesized cDNA into vectors, such as λZAP; (3) spreading the cDNAlibrary; and (4) conducting colony hybridization or plaque hybridizationas described above. Alternatively, cDNA can also be prepared by PCR.

[0045] The present invention includes DNAs encoding proteinsfunctionally equivalent to the RISBZ protein of SEQ ID NO: 2, 5, or 7.Herein, the term “functionally equivalent to the RISBZ protein” meansthat the object protein has the biological function equivalent to thoseof RISBZ protein of SEQ ID NO: 2, 5, or 7, such as the function ofbinding to GCN4 motif and/or regulating the expression of rice seedstorage proteins. The rice seed storage proteins include, for example,rice glutelins.

[0046] Examples of such DNAs include those encoding mutants,derivatives, alleles, variants, and homologues comprising the amino acidsequence of. SEQ ID NO: 2, 5, or 7 wherein one or more amino acids aresubstituted, deleted, added, and/or inserted.

[0047] Examples of methods for preparing a DNA encoding a proteincomprising altered amino acids well known to those skilled in the artinclude the site-directed mutagenesis (Kramer, W. and Fritz, H. -J.,Oligonucleotide-directed construction of mutagenesis via gapped duplexDNA. Methods in Enzymology, 154: 350-367, 1987). The amino acid sequenceof a protein may also be mutated spontaneously due to the mutation of anucleotide sequence. A DNA encoding proteins having the amino acidsequence of a natural RISBZ protein (SEQ ID NOs: 2, 5, or 7) wherein oneor more amino acids are substituted, deleted, and/or added are alsoincluded in the DNA of the present invention, so long as they encode aprotein functionally equivalent to the natural RISBZ protein.Additionally, nucleotide sequence mutants that do not give rise to aminoacid sequence changes in the protein (degeneracy mutants) are alsoincluded in the DNA of the present invention. The numbers of nucleotidemutations in the object DNA at amino acid level is typically 100 aminoacids or less, preferably 50 amino acids or less, more preferably 20amino acids or less, and most preferably 10 amino acids or less (forexample, amino acids or less or 3 amino acids or less).

[0048] Whether or not a certain DNA codes for a protein having thefunction of binding to the GCN4 motif can be determined by, for example,gel shift assay usually used by those skilled in the art. Morespecifically, this assay can be carried out as follows: First, thedetected DNA is incorporated into a vector so that its gene productforms a fused protein with GST and the vector is allowed to express thefused protein. The expression product is purified using GST as anindicator followed by mixing with a labeled DNA probe containing theGCN4 motif. This mixed solution is analyzed by electrophoresis usingnondenaturing acrylamide gel. Binding activity can then be evaluatedbased on the locations of the detected bands on the gel.

[0049] In addition, whether or not a certain DNA codes for a proteinhaving the function of activating expression of rice seed storageprotein can be determined by, for example, a reporter assay. Morespecifically, this assay can be carried out as follows. First, a vectoris constructed so that a reporter gene is connected to and downstream ofthe promoter of rice seed storage protein. This vector and a vector thatexpresses the gene product of a test DNA are introduced into the cellsfor the reporter assay, and the transcription activity of the test DNAgene product is evaluated by measuring the activity of the reporter geneproduct. An example of the promoter of rice seed storage protein thatcan be used for the reporter assay is the rice glutelin gene promoter.There are no particular restrictions to the reporter gene provided itsexpression can be detected, and any reporter gene that are usually usedin various assay systems by those skilled in the art, can be used. Apreferable example of the reporter gene is the β-glucuronidase (GUS)gene.

[0050] A DNA encoding a protein functionally equivalent to the RISBZprotein set forth in SEQ ID NO: 2, 5, or 7 can be produced by, forexample, methods well known to those skilled in the art including:methods using hybridization techniques (Southern, E. M., Journal ofMolecular Biology, Vol. 98, 503, 1975); and polymerase chain reaction(PCR) techniques (Saiki, R. K. et al. Science, 230, 1350-1354, 1985;Saiki, R. K. et al. Science, 239, 487-491, 1988). It is routine for aperson skilled in the art to isolate a DNA with high homology to theRISBZ gene from rice and so forth using the RISBZ gene (SEQ ID NO: 1, 3,4, or 6) or parts thereof as a probe, and oligonucleotides hybridizingspecifically to the gene as a primer. Such a DNA encoding a proteinfunctionally equivalent to the RISBZ protein, isolable by hybridizationtechniques or PCR techniques, is included in the DNA of this invention.

[0051] Hybridization reactions to isolate such DNAs are preferablyconducted under stringent conditions. Stringent hybridization conditionsof the present invention include conditions such as: 6 M urea, 0.4% SDS,and 0.5×SSC; and those which yield a similar stringency to theconditions. DNAs with higher homology are expected to be isolatedefficiently when hybridization is performed under conditions with higherstringency, for example, 6 M urea, 0.4% SDS, and 0.1×SSC. These DNAsisolated under such conditions are expected to encode a protein having ahigh amino acid level homology with RISBZ protein (SEQ ID NO: 2, 5, or7). Herein, high homology means an identity of at least 50% or more,more preferably means an identity of at least 70% or more, and mostpreferably means an identity of at least 90% or more (e.g., 95% or more)throughout the entire amino acid sequence. The degree of sequenceidentity can be determined by FASTA search (Pearson W. R. and D. J.Lipman Proc. Natl. Acad. Sci. USA. 85:2444-2448, 1988) or BLAST search.

[0052] The DNA of the present invention can be used, for example, toprepare recombinant proteins and to produce transgenic plants asdescribed above.

[0053] A recombinant protein is usually prepared by inserting a DNAencoding a protein of the present invention into an appropriateexpression vector, introducing the vector into an appropriate cell,culturing the transformed cells, and purifying expressed proteins. Arecombinant protein can be expressed as a fusion protein with otherproteins so as to be easily purified, for example, as a fusion proteinwith maltose binding protein in Escherichia coli (New England Biolabs,USA, vector pMAL series), as a fusion protein withglutathione-S-transferase (GST) (Amersham Pharmacia Biotech, vector pGEXseries), or tagged with histidine (Novagen, pET series). The host cellis not limited so long as the cell is suitable for expressing therecombinant protein. It is possible to utilize, for example, yeast,plant, insect cells or various other animal cells besides theabove-described E. coli. A vector can be introduced into a host cell bya variety of methods known to one skilled in the art. For example, atransformation method using calcium ions (Mandel, M. and Higa, A.Journal of Molecular Biology, 53, 158-162,1970; Hanahan, D. Journal ofMolecular Biology, 166, 557-580, 1983) can be used to introduce a vectorinto E. coli. A recombinant protein expressed in the host cells can bepurified and recovered from the host cells or the culture supernatantthereof by known methods in the art. When a recombinant protein isexpressed as a fusion protein with maltose binding protein or otherpartners, the recombinant protein can be easily purified via affinitychromatography.

[0054] The resulting protein can be used to prepare an antibody thatbinds to the protein. For example, a polyclonal antibody can be preparedby immunizing immune animals, such as rabbits, with a purified proteinof the present invention or its portion, collecting blood after acertain period, and removing clots. A monoclonal antibody can beprepared by fusing myeloma cells with the antibody-forming cells ofanimals immunized with the above protein or its portion, isolating amonoclonal cell expressing a desired antibody (hybridoma), andrecovering the antibody from the cell. The antibody thus obtained can beutilized to purify or detect a protein of the present invention.Accordingly, the present invention includes antibodies that bind toproteins of the invention.

[0055] A plant transformant expressing DNAs of the present invention canbe created by inserting a DNA encoding a protein of the presentinvention into an appropriate vector, introducing this vector into aplant cell, and then, regenerating the resulting transformed plant cell.

[0056] On the other hand, a plant transformant in which the expressionof the DNA of the present invention is suppressed can be created using aDNA that suppresses the expression of a DNA encoding a protein of thepresent invention: wherein the DNA is inserted into an appropriatevector, the vector is introduced into a plant cell, and then, theresulting transformed plant cell is regenerated. The phrase “suppressionof expression of a DNA encoding a protein of the present invention”includes suppression of gene transcription as well as suppression oftranslation to protein. Furthermore, it also includes the completeinability of expression of DNA as well as reduction of expression.

[0057] The expression of a specific endogenous gene in plants can besuppressed by methods utilizing antisense technology conventional to theart. Ecker et al. were the first to demonstrate the antisense effect ofan antisense RNA introduced by electroporation into plant cells by usingthe transient gene expression method (J. R. Ecker and R. W. Davis Proc.Natl. Acad. Sci. USA 83: 5372, 1986). Thereafter, the target geneexpression was reportedly reduced in tobacco and petunias by expressingantisense RNAs (A. R. van der Krol et al. Nature 333: 866, 1988). Theantisense technique has now been established as a means of suppressingtarget-gene expression in plants.

[0058] Multiple factors cause antisense nucleic acid to suppress thetarget-gene expression. These include the following: inhibition oftranscription initiation by triple strand formation; suppression oftranscription by hybrid formation at the site where the RNA polymerasehas formed a local open loop structure; transcription inhibition byhybrid formation with the RNA being synthesized; suppression of splicingby hybrid formation at the junction between an intron and an exon;suppression of splicing by hybrid formation at the site of spliceosomeformation; suppression of mRNA translocation from the nucleus to thecytoplasm by hybrid formation with mRNA; suppression of splicing byhybrid formation at the capping site or at the poly(A) addition site;suppression of translation initiation by hybrid formation at the bindingsite for the translation initiation factors; suppression of translationby hybrid formation at the site for ribosome binding near the initiationcodon; inhibition of peptide chain elongation by hybrid formation in thetranslated region or at the polysome binding sites of mRNA; andsuppression of gene expression by hybrid formation at the sites ofinteraction between nucleic acids and proteins. These factors suppressthe target gene expression by inhibiting the process of transcription,splicing, or translation (Hirashima and Inoue, “Shin Seikagaku JikkenKoza (New Biochemistry Experimentation Lectures) 2, Kakusan (NucleicAcids) IV, Idenshi No Fukusei To Hatsugen (Replication and Expression ofGenes),” Nihon Seikagakukai Hen (The Japanese Biochemical Society),Tokyo Kagaku Dozin, pp. 319-347, (1993)).

[0059] An antisense sequence of the present invention can suppress thetarget gene expression by any of the above mechanisms. In oneembodiment, if an antisense sequence is designed to be complementary tothe untranslated region near the 5′ end of the gene's mRNA, it willeffectively inhibit translation of a gene. It is also possible to usesequences complementary to the coding regions or to the untranslatedregion on the 3′ side. Thus, the antisense DNA used in the presentinvention includes a DNA having antisense sequences against both theuntranslated regions and the translated regions of the gene. Theantisense DNA to be used is connected downstream of an appropriatepromoter, and, preferably, a sequence containing the transcriptiontermination signal is connected on the 3′ side. The DNA thus preparedcan be transfected into the desired plant by known methods. The sequenceof the antisense DNA is preferably a sequence complementary to theendogenous gene of the plant to be transformed or a part thereof, but itneed not be perfectly complementary so long as it can effectivelyinhibit the gene expression. The transcribed RNA is preferably 90% ormore, and most preferably 95% or more complementary to the transcribedproducts of the target gene. The complementary of sequences can bedetermined by the above-described search methods. In order toeffectively inhibit the expression of the target gene by means of anantisense sequence, the antisense DNA should be at least 15 nucleotideslong or more, preferably 100 nucleotides long or more, and still morepreferably 500 nucleotides long or more. The antisense DNA to be used isgenerally shorter than 5 kb, and preferably shorter than 2.5 kb.

[0060] DNA encoding ribozymes can also be used to suppress theexpression of endogenous genes. A ribozyme means an RNA molecule thathas catalytic activities. There are many ribozymes having variousactivities. Research on the ribozymes as RNA cleaving enzyme has enabledthe design of a ribozyme that site-specifically cleaves RNA. While someribozymes of the group I intron type or the M1RNA contained in RNasePconsist of 400 nucleotides or more, others belonging to the hammerheadtype or the hairpin type have an activity domain of about 40 nucleotides(Makoto Koizumi and Eiko Ohtsuka Tanpakushitsu Kakusan Kohso (Nucleicacid, Protein, and Enzyme) 35: 2191, 1990).

[0061] The self-cleavage domain of a hammerhead type ribozyme cleaves atthe 3′ side of C15 of the sequence G13U14C15. Formation of a nucleotidepair between U14 and A at the ninth position is considered important forthe ribozyme activity. It has been shown that the cleavage also occurswhen the nucleotide at the 15th position is A or U instead of C (M.Koizumi et al. FEBS Lett. 228: 225, 1988). If the substrate binding siteof the ribozyme is designed to be complementary to the RNA sequencesadjacent to the target site, one can create a restriction-enzyme-likeRNA cleaving ribozyme which recognizes the sequence UC, UU, or UA withinthe target RNA (M. Koizumi et al. FEBS Lett. 239: 285, 1988; MakotoKoizumi and Eiko Ohtsuka Tanpakushitsu Kakusan Kohso (Protein, Nucleicacid, and Enzyme), 35: 2191, 1990; M. Koizumi et al. Nucleic Acids Res.17: 7059, 1989). For example, in the coding region of the RISBZ gene(SEQ ID NO: 1, 3, 4, or 6), there are pluralities of sites that can beused as the ribozyme target.

[0062] The hairpin-type ribozyme is also useful in the presentinvention. A hairpin-type ribozyme can be found, for example, in theminus strand of the satellite RNA of tobacco ringspot virus (J. M.Buzayan, Nature 323: 349,1986). This ribozyme has also been shown totarget-specifically cleave RNA (Y. Kikuchi and N. Sasaki (1992) NucleicAcids Res. 19: 6751; Yo Kikuchi (1992) Kagaku To Seibutsu (Chemistry andBiology) 30: 112).

[0063] The ribozyme designed to cleave the target is fused with apromoter, such as the cauliflower mosaic virus ³⁵S promoter, and with atranscription termination sequence, so that it will be transcribed inplant cells. If extra sequences have been added to the 5′ end or the 3′end of the transcribed RNA, the ribozyme activity can be lost. In thiscase, one can place an additional trimming ribozyme, which functions incis to perform the trimming on the 5′ or the 3′ side of the ribozymeportion, in order to precisely cut the ribozyme portion from thetranscribed RNA containing the ribozyme (K. Taira et al. (1990) ProteinEng. 3: 733; A. M. Dzaianott and J. J. Bujarski (1989) Proc. Natl. Acad.Sci. USA 86: 4823; C. A. Grosshands and R. T. Cech (1991) Nucleic AcidsRes. 19: 3875; K. Taira et al. (1991) Nucleic Acid Res. 19: 5125).Multiple sites within the target gene can be cleaved by arranging thesestructural units in tandem to achieve greater effects (N. Yuyama et al.,Biochem. Biophys. Res. Commun. 186: 1271 (1992)). By using suchribozymes, it is possible to specifically cleave the transcriptionproducts of the target gene in the present invention, therebysuppressing the expression of the gene.

[0064] Endogenous gene expression can also be suppressed byco-suppression through the transformation by DNA having a sequenceidentical or similar to the target gene sequence. “Co-suppression”refers to the phenomenon in which, when a gene having a sequenceidentical or similar to the target endogenous gene sequence isintroduced into plants by transformation, expression of both theintroduced exogenous gene and the target endogenous gene becomessuppressed. Although the detailed mechanism of co-suppression isunknown, it is frequently observed in plants (Curr. Biol. 7: R793, 1997,Curr. Biol. 6: 810, 1996). For example, if one wishes to obtain a plantbody in which the RISBZ gene is co-suppressed, the plant in question canbe transformed via a vector DNA designed so as to express the RISBZ geneor DNA having a similar sequence to select a plant having the RISBZmutant character, for example, a plant with modified expression level ofstorage proteins in seeds, among the resultant plants. The gene to beused for co-suppression need not be identical to the target gene, but itshould have at least 70% or more sequence identity, preferably 80% ormore sequence identity, and more preferably 90% or more (e.g., 95% ormore) sequence identity. Sequence identity can be determined by usingthe above-described search.

[0065] In addition, endogenous gene expression in the present inventioncan also be suppressed by transforming the plant with a gene encoding aprotein having the dominant negative phenotype of the expression productof the target gene. “A DNA encoding a protein having the dominantnegative phenotype” as used herein means a DNA encoding a protein, whichupon expression, can eliminate or reduce the activity of the proteinencoded by endogenous gene inherent to the plant. An example thereof isa DNA that codes for a peptide having GCN4 binding ability and having notranscription activating domain of the protein of the present invention(for example, the peptide missing the 1st to 40th amino acids of theamino acid sequence of SEQ ID NO: 2 or a peptide of other proteinscorresponding thereto).

[0066] The vector used to transform plant cells is not particularlyrestricted as long as it is capable of expressing an inserted gene inthe cells. For example, a vector having a promoter for performingconstitutive gene expression in plant cells (e.g., the ³⁵S promoter ofcauliflower mosaic virus), or a vector having a promoter that isinductively activated by an external stimulus can be used. In addition,a promoter that guarantees tissue-specific expression can also besuitably used. Examples of tissue-specific promoters include a promoterof glutelin gene (Takaiwa, F. et al., Plant Mol. Biol. 17: 875-885,1991) or a promoter of the RISBZ1 of the present invention for theexpression in the seeds of rice plants, and a promoter of glycinin genefor the expression in the seeds of leguminous crops such as kidneybeans, broad beans and green peas or oil seed crops such as peanuts,sesame seeds, rape seeds, cottonseeds, sunflower seeds and safflowerseeds, or a promoter of the major storage protein of each of the abovecrops such as a promoter of phaseolin gene in the case of kidney beans(Murai, N. et al., Science 222: 476-482, 1993) or a promoter of thegluciferrin gene in the case of rape seed (Rodin, J. et al., Plant Mol.Biol. 20: 559-563, 1992), a promoter of the patatin gene (Rocha-Sosa, M.et al., EMBO J. 8: 23-29, 1989) for the expression in the root tuber ofpotatoes, a promoter of the sporamin gene for the expression in the roottuber of sweet potatoes (Hattori, T. and Nakamura, K., Plant Mol. Biol.11: 417-426, 1988), and a promoter of the ribulose-1,5-bisphosphatedecarboxylase gene for the expression in the leaves of spinach and othervegetables (Orozco, B. M. and Ogren, W. L., Plant Mol. Biol. 23:1129-1138, 1993).

[0067] The plant cell to which a vector is introduced used hereinincludes various forms of plant cells, such as cultured cellsuspensions, protoplasts, leaf sections, and callus.

[0068] A vector can be introduced into plant cells by known methods,such as the polyethylene glycol method, electroporation,Agrobacterium-mediated transfer, and particle bombardment. Plants can beregenerated from transformed plant cells by known methods depending onthe type of the plant cell (Toki et al., (1995) Plant Physiol.100:1503-1507). For example, transformation and regeneration methods forrice plants include: (1) introducing genes into protoplasts usingpolyethylene glycol and regenerating the plant body (suitable for indicarice cultivars) (Datta, S. K. (1995) in “Gene Transfer To Plants”,Potrykus I and Spangenberg Eds., pp66-74); (2) introducing genes intoprotoplasts using electric pulse, and regenerating the plant body(suitable for japonica rice cultivars)(Toki et al (1992) Plant Physiol.100, 1503-1507); (3) introducing genes directly into cells by theparticle bombardment, and regenerating the plant body (Christou et al.(1991) Bio/Technology, 9: 957-962); (4) introducing genes usingAgrobacterium, and regenerating the plant body (Hiei et al. (1994) PlantJ. 6: 271-282); and so on. These methods are already established in theart and are widely used in the technical field of the present invention.Such methods can be suitably used for the present invention.

[0069] Once a transformed plant with the DNA of the present inventionintegrated into the genome is obtained, it is possible to gain progeniesfrom that plant body by sexual or vegetative propagation. Alternatively,plants can be mass-produced from breeding materials (for example, seeds,fruits, ears, tubers, tubercles, tubs, callus, protoplast, etc.)obtained from the plant, as well as progenies or clones thereof. Plantcells transformed with the DNA of the present invention, plant bodiesincluding these cells, progenies and clones of the plant, as well asbreeding materials obtained from the plant, its progenies and clones,are all included in the present invention. The plant body of the presentinvention is preferably a monocotyledon, more preferably a plant of thePoaceae, and most preferably a rice plant.

[0070] In addition, the present invention provides a plant body in whicha foreign gene product has been highly expressed using the RISBZ gene ofthe present invention. The plant body of the present invention has inits genome a DNA construct in which the DNA of the present invention isoperably connected downstream of an expression control region, and a DNAconstruct in which a foreign gene is operably connected downstream of anexpression control region having a target sequence.

[0071] The DNA of the present invention or a foreign gene being“operably connected” downstream of an expression control region meansthat the DNA of the present invention or a foreign gene binds to anexpression control region so as to induce the expression of the DNA ofthe present invention or a foreign gene by the binding of atranscription factor to the expression control region.

[0072] The target sequence refers to a DNA sequence to which the RISBZprotein of the present invention, which is a transcription factor,binds, and is preferably a DNA sequence that contains the GCN4 motif orG/C box. Examples of the GCN4 motif include the sequences shown belowwhich have been found in various genes:

[0073] *GCN4 Motif (name of gene containing GCN4 motif)GCTGAGTCATGA/(GluB-1) SEQ ID NO: 8 CATGAGTCACTT/(GluA-1) SEQ ID NO: 9AGTGAGTCACTT/(GluA-3) SEQ ID NO: 10 GGTGAGTCATAT/(LMWG) SEQ ID NO: 11GGTGAGTCATGT/(Hordein) SEQ ID NO: 12 GATGAGTCATGC/(Gliadin) SEQ ID NO:13 AATGAGTCATCA/(Secalin). SEQ ID NO: 14

[0074] Preferable GCN4 motif sequences for use as target sequencesinclude “GCTGAGTCATGA/SEQ ID NO: 8”, GATGAGTCATGC/SEQ ID NO: 13” and“AATGAGTCATCA/SEQ ID NO: 14”. Specific examples of a G/C box include thesequence, “AGCCACGTCACA/SEQ ID NO: 15”. Sequences in which the aboveGCN4 motif or G/C box is repeated in tandem are also included in thetarget sequence of the present invention, and a preferable example is asequence in which the GCN4 motif or G/C box are repeated in tandem fourtimes.

[0075] Examples of foreign genes include genes coding for antibodies,enzymes, and physiologically active peptides.

[0076] Moreover, the present invention provides a method of producing aplant body in which a foreign gene product is highly expressed using theRISBZ gene of the present invention. Examples of the methods forproducing the plant body include a method of crossing “a plant bodyhaving a DNA construct in its genome, in which the DNA of the presentinvention is operably connected downstream of an expression controlregion,” and “a plant body having a DNA construct in its genome, inwhich a foreign gene is operably connected downstream of an expressioncontrol region having the target sequence of the protein of the presentinvention.”

[0077] The above-described “DNA construct in which the DNA of thepresent invention is operably connected downstream of an expressioncontrol region,” and “the DNA construct in which a foreign gene isoperably connected downstream of an expression control region having atarget sequence” can be introduced into the plant genome by aconventional method by those skilled in the art, such as a method thatuses the above-mentioned agrobacterium.

[0078] In addition, crossing of plant bodies can be carried out by aconventional method for those skilled in the art. For example, in orderto prevent self-propagation, only the pollen is sterilized bydemasculating using the tip shearing method on the day of crossing or bydemasculating using hot water on the day of crossing to shake pollinatethe ear of the pollen mother.

BRIEF DESCRIPTION OF THE DRAWINGS

[0079]FIG. 1 is a drawing representing a genealogical tree based on thehomology of the amino acid sequence of RISBZ protein and O2-like bZIPprotein. The entire amino acid sequences of these proteins are comparedto understand the similarity and the evolutionary relationship of theseproteins.

[0080]FIG. 2 compares the amino acid sequences of RISBZ protein andO2-like bZIP protein. Outline letters on a black background shows theamino acids that retained 50% or more. The presumed nuclear migrationsignal (NLSA: SV40-like motif) (Varagona, M. J. et al., Plant Cell 4:1213-1227, 1992) and the serine-rich phosphorylation sites are indicatedwith double lines and broken lines, respectively. The bold linesindicate the basic domain, which has a two-factor nuclear migrationsignal (NLSB) structure. Downward arrows indicate the leucine repeats.The primer used for the production of the rice bZIP probe was designedbased on the amino acid sequences indicated by rightward and leftwardarrows. BLZ1 (Vicente-Carbojos, J. et al., Plant J. 13: 629-640, 1998)and BLZ2 (Onate, L. et al., J. Biol. Chem. 274: 9175-9182, 1999)represent O2-like bZIP proteins isolated from barley, O2 (Hartings, H.et al., EMBO J. 8: 2795-2801, 1989) and OHP1 (Pysh, L. D. et al., PlantCell 5: 227-236, 1993) from maize, SPA from wheat (Albani D. et al.,Plant Cell9: 171-184, 1997), O2-sorg from sorghum (Pirovano, L. et al.,Plant Mol. Biol. 24: 515-523, 1994), and O2-coix from adlay (Vettore, A.L. et al., Plant Mol. Biol. 36: 249-263, 1998).

[0081]FIG. 3 is a continuation of FIG. 2.

[0082]FIG. 4 shows the structure of a gene that codes for O2-like bZIPprotein. The structures of the intron/exon region of the BLZ1 gene ofbarley and the Opaque2 gene of maize (O2) (Hartings, H. et al., EMBO J.8: 2795-2801, 1989), sorghum (O2-sorg) and adlay (O2-coix) are shown.The thick bars and thin lines represent exons and introns, respectively.The numbers indicate the number of nucleotides of the exons and introns.

[0083]FIG. 5 is a photograph representing the result of a Northern blotshowing the transcription patterns of the RISBZ genes. Northern blottinganalysis was performed on the whole RNA extracted from the root,seedling, and maturing seeds (5, 10, 15, 20, and 30 DAF) using a uniquenucleotide sequence of a region downstream of the bZIP domain for theprobe. In order to compare transcription patterns, the analysis was alsoconducted using the. GluB-1 gene-coding region as the probe. The stainedimages of 25S rRNA obtained using ethidium bromide are shown as acontrol.

[0084]FIG. 6 represents the results of histological analysis of theRISBZ1 promoter/GUS reporter gene in a transformed rice plant.

[0085] (A) is a schematic drawing of the RISBZL promoter/GUS reportergene. (a) and (b) show the sequence from the −1674^(th) to +4^(th)nucleotides counting from the transcription initiation point of theRISBZ1 gene and the sequence from the −1674^(th) to +213^(th) gene thatcontains uORF, respectively, both connected to the GUS reporter gene ona binary vector. (c) shows the GluB1 promoter (−245 to +18) sequencebinding to the GUS reporter gene on a plasmid vector.

[0086] (B) are photographs showing the expression of GUS reporter genein a seed during the maturation process. After cutting the seed (10 DAF)of a rice plant, into which the reporter gene was introduced, in thelongitudinal direction, the cut seed was immersed in X-gluc solution andincubated at 37° C. EN indicates the endosperm, while EM indicates theembryo.

[0087] (C) is a graph showing the GUS activity of a seed extract of atransformed rice plant. 15 DAF seeds were used for analysis. Thepromoter structures of the introduced genes are as shown in (a) and (b)of (A), respectively. Vertical lines indicate the mean value. MUrepresents 4-methylumbelliferone.

[0088]FIG. 7 shows photographs of gel electrophoretic patterns asdetermined from a methylation interference experiment for identifyingthe RISBZ1 protein-binding site on the GluB1 promoter. Each of thestrands (top and bottom) of the promoter fragment of the GluB1 gene(−245 to +18) was labeled. After partially methylating each strand, theywere incubated with GST-RISBZ1 protein, the fragments that did not bindto the protein and the fragment that bound to the protein were eachcollected and subjected to electrophoresis after chemically cleaved bypiperidine. The sites (indicated by asterisks) that were not cleaved bypiperidine were only found in the GCN4 motif.

[0089]FIG. 8 shows the result of electrophoresis in gel shift analysisto investigate the binding capability of RISBZ1 protein to the GCN4motif.

[0090] (A) shows 21-bp DNA fragments that contain the GCN4 motif of aWILD:GluB-1 promoter sequence (−175 to −155) of an oligonucleotide usedas the probe and competitor. M1 to M7 are a series of 21-bp DNAfragments that were mutated every 3 bp. The GCN4 motif is underlined.

[0091] (B) through (F) show the results of gel shift analysis of theGST-RISBZ fused protein. A 21-bp DNA fragment (WILD) was added as theprobe. (B) is for GST-RISBZ1, (C) for GST-RISBZ2, (D) for GST-RISBZ3,(E) for GST-RISBZ4, and (F) for GST-RISBZ5. The competitor was added toa stoichiometric ratio of 100 times or more against the probe. Lane 1:No protein; Lane 2: No competitor; and Lanes 3 to 10: With Competitor(wild type (W) and M1 to M7).

[0092]FIG. 9 represents heterodimer forming ability of RISBZ1 with otherRISBZ proteins.

[0093] (A) shows the vector structure used as the in vitrotranscription/translation reaction template. The vectors contain DNAcoding for full-length RISBZ1 protein, short-form RISBZ2 protein(sRISBZ2: 218 to 329), or short-form RISBZ3 protein (sRISBZ3: 126 to237).

[0094] (B) shows photographs of gel electrophoretic patternsrepresenting the results of a DNA binding assay. In lanes 2, 4, 6, and8, DNA complexes that bound to the full length or short-form proteinwere detected. In lanes 3 and 7, DNA complexes that bound to theheterodimer of full length RISBZ1 protein and short-form protein weredetected.

[0095]FIG. 10 shows the results of identification of thetranscription-activating domain determined by transient analysis.

[0096] (A) shows the structure of the reporter and effector plasmid. AGUS gene in which 9 copies of GAL4-DNA binding sites and CaMV35S corepromoter sequence are linked was used for the reporter. The effectorplasmid contained DNA coding for a protein in which the GAL4 DNA bindingdomain was linked to the N-terminal side of truncated RISBZ1 protein.

[0097] (B) is a graph showing GUS activity when the reporter andeffector plasmid were used.

[0098]FIG. 11 shows the hydropathy patterns of the N-terminal region ofRISBZ1 (WT) and mutant RISBZ1 (M1 to 8) proteins determined by theformula of Kyte and Doolittle (Kyte, J. and Doolittle, R. F. J., Mol.Biol. 157: 105-132, 1982). Positive values indicate hydropathy.

[0099]FIG. 12 schematically shows the transcription activity measurementsystem of RIZBZ1 using GUS activity as the indicator, photographs ofNorthern blot analysis, and a graph showing GUS activity measurementresults. The ordinate of the graph represents GUS activity that is theindicator of the strength of the transcription activity of eachtranscription factor.

[0100]FIG. 13 is a graph showing the recognition sequences oftranscription factors RISBZ1, Opaque2, SPA, and RISBZ3 (RITA1). Theordinate of the graph represents GUS activity that is the indicator ofthe strength of the transcription activity of each transcription factor.The sequences used in the experiment are shown below the graph.

[0101]FIG. 14 is a graph showing the transcription activating ability ofthe RISBZ1 of the present invention relative to GCN4 motifs originatingin various genes. The ordinate of the graph represents GUS activity,which is the indicator of the strength of the transcription activity ofeach transcription factor. The nucleotide sequences of the GCN4 motifsused in the experiment are shown below the graph.

MODE FOR CARRYING OUT THE INVENTION

[0102] The present invention will be described in more detail below withreference to Examples, but is not to be construed as being limitedthereto.

EXAMPLE 1 Isolation of cDNA Clones Encoding the bZIP TranscriptionFactor From Seed cDNA Libraries

[0103] Fourteen-day leaves and roots of rice plant (Oryza sativa L. c.v. Mangetumochi) cultivated by hydroponics were frozen in liquidnitrogen and kept at −80° C. until use. Maturing rice seeds werecollected from rice plants cultivated in the fields.

[0104] Using oligonucleotide primers designed from highly conservedamino acid sequences (SNRESA and KVKMAED) within the bZIP domain of theOpaque 2 (O2)-like protein, RT-PCR was performed by using poly(A)⁺ mRNAas a template, which was prepared from the rice seeds. From poly (A) RNAextracted from seeds at 6 to 16 days after flowering (DAF) (Takaiwa F.et al. Mol. Gen. Genet. 208: 15-22, 1987), single-stranded cDNA wassynthesized by reverse transcription using oligo(dT)₂₀ as a primer andSuperscript reverse transcriptase (Gibco BRL, Paisly, UK). Next, cDNAwas amplified using a pair of primers (5′-TCC AAC/T A/CGI GAA/G A/TCIGC-3′; SEQ ID NO: 16, and 5′-GTC CTC C/TGC CAT CTT CAC CTT-3′; SEQ IDNO: 17). These primers were designed based on highly conserved aminoacid sequences within the bZIP-type transcription factors that wereexpressed in cereal seeds. After dissolving the single-stranded cDNA ina PCR reaction mixture containing 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl₂,50 mM KCl, 0.01% (w/v) gelatin, 200 μM dNTPs, 1 μM oligonucleotideprimers, TaqI polymerase was added to the mixture and the resultingmixture was incubated in a thermal cycler at 94° C. for 5 min. cDNA wasthen synthesized and amplified by three-cycle PCR (for 1 min at 94° C.,for 1 min at 40° C., and then for 2 mins at 72° C.) followed by 30-cyclePCR (for 1 min at 94° C., for 1 min at 55° C., and then for 2 mins at72° C.). The amplified DNA fragment was cloned into a TA cloning vector(pCR2.1; Invitrogen), and subjected to sequencing by using the ABI PRISMdye terminator sequence system. The reaction products were analyzed byABI PRISM 310 Genetic Analyzer (Perkin Elmer-Applied Biosystems) todetermine the nucleotide sequences of at least 50 clones. The obtainednucleotide sequence data was analysed and searched on databases by usingthe GENETYX and BLAST algorisms. As a result, five distinct DNAfragments with 213-bp were found. Two of these were identical to thebZIP domain sequences of REB (Izawa T. et al. Plant Cell 6: 1277-1287,1994) and the RITA1 (Nakase M. et al. Plant Mol. Biol. 33: 513-522,1997). Using the five DNA fragments with 213-bp as primers, a cDNAlibrary was prepared from RNA of maturing (6-16DAF) seeds (ZAPII;STRATAGENE) This was then screened to obtain their full-length cDNAscorresponding to each of the fragments under high stringent conditions.[α-³²P]-dCTP was incorporated into the DNA fragments by random priming(Amersham Pharmacia Biotech) and the resulting fragments were used asprobes. As a pre-hybridisation solution, a mixture containing 5×SSC, 5×Denhard's solution, 0.1% SDS, 50% formamide, 100 μg/ml salmon sperm DNAwas used. After hybridization, filters were washed once at 55° C. with amixture consisting of 2×SSC and 0.1% SDS, and then twice at 55° C. witha mixture consisting of 0.1×SSC and 0.1% SDS.

[0105] Based on the homologies to each nucleotide sequence, the cDNAclones obtained were termed as RISBZ1 (rice seed b-Zipper 1) (SEQ ID NO:1), RISBZ2, RISBZ3, RISBZ4 (SEQ ID NO: 4), and RISBZ5 (SEQ ID NO: 6).Among them, RISBZ2 and RISBZ3 were identical to REB (Izawa T. et al.Plant Cell 6: 1277-1287, 1994) and RITAL (Nakase M. et al. Plant Mol.Biol. 33: 513-522, 1997), respectively, which have previously beenisolated from cDNA libraries of seeds and leaves.

EXAMPLE 2 Identification of RISBZ cDNA

[0106] The newly identified RISBZ cDNAs (RISBZ1, RISBZ4, and RISBZ5)were characterized in detail as described below. RISBZ1 cDNA was thelongest, which had 1742 bp in length excluding poly(A), and contained areading frame encoding 436 amino acids that had 46,491 Dal of anestimated molecular weight. RISBZ4 and RISBZ5 have reading framesencoding 278 and 295 amino acids; their estimated molecular weights are29,383 Dal and 31,925 Dal respectively.

[0107] RISBZ1 mRNA has a longer leader sequence (245 bases long) thanaverage leader sequences. Interestingly, a small open reading frame,encoding 31 amino acid residues, was found within the leader sequence inthe upstream of the actual initiation codon of the RISBZ1 protein.Similar small upstream open reading frames (UORF) have previously beenfound in maize Opaque 2 (O2) (Hartings H. et al. EMBO J. 8: 2795-2801,1989), wheat SPA (Albani D. et al. Plant Cell 9: 171-184, 1997), andbarley BLZ1 and BLZ2 (Vincente-Carbojos J. et al. Plant J. 13: 629-640,1998; Onate L. et al. J. Biol. Chem. 274: 9175-9182, 1999), but theseuORFs have little homology with each other. It has previously beenreported that uORF of the maize O2 mRNA is involved in translationalcontrol. uORF was found only in RISBZ1 mRNA but not in other RISBZ mRNA.

[0108] The flanking sequence of the initiation codon is GCAATGG. Thissequence coincided with eukaryotic translational initiation sequence,c(a/c) (A/G) (A/C)cAUGGCG, derived from monocotyledonous plants. Therewere 100 bps between the initiation codon and uORF. The open readingframe encoding RISBZ1 had two identical termination codons (TAG). Therewere 229 bps between the termination codon and poly (A) sequence. Thepolyadenylation signal sequence (AATATA) was found in the region at −19to −24 from the site to which poly(A) was added.

[0109] RISBZ1 is closely related to rice REB (Nakase M. et al. PlantMol. Biol. 33: 513-522, 1997), maize OHP-1 and OHP-2 (Pysh L. D. et al.Plant Cell 5: 227-236, 1993), and barley BLZ1 (Vincente-Carbojos J. etal. Plant J. 13: 629-640, 1998) (FIG. 1), and showed the homologies of48.2% (rice REB), 45.7% (barley BLZ1), and 46.6% (maize OHPL),respectively, at the amino acid level. Furthermore, these bZIP domainswere highly conserved (73.7% to 76.3%). At the amino acid level, thehomologies of RITA1 (RISBZ3) with RISBZ4 and RISBZ5 were 88.8% and 47.6%respectively. By contrast, the homology of RISBZ4 with RISBZ5 was 48.2%.RISBZ3, RISBZ4, and RISBZ5 comprise a unique group among the O2-liketranscription factors that were previously reported. Furthermore, thefive RISBZ cDNAs isolated from the seed cDNA library could be classifiedinto two groups based upon the amino acid homology (FIG. 1). The RISBZ3,RISBZ4, and RISBZ5 lacked the N- and C-terminal regions present inRISBZ1 and RISBZ2, and their sizes reduced about 100 to 150 amino acidresidues compared with those of RISBZ1 and RISBZ2 (FIGS. 2 and 3).

[0110] RISBZ1 and RISBZ2 were rich in proline residues at theirN-terminal region, which lacked in other RISBZ proteins (FIGS. 2 and 3).RISBZ1 and RISBZ2 were also rich in acidic amino acids at the peripheralregion of the 60^(th) amino acid residue from their N-termini and at theintermediate region located in the upstream of their bZIP domains. Theseproline-rich or acidic amino acid-rich regions were found in otherO2-like transcription factors.

[0111] Since serine-rich sequence (SGSS) was found in the region rangingfrom 207^(th) to 210^(th) residues of RISBZ1, the protein was consideredto be a target sequence of casein kinase II (Hunter T. and Karin M. Cell70: 375-387, 1992) (FIGS. 2 and 3) Similar sequence (SSSS) was alsofound in RISBZ2. However, it was missing in the other RISBZ proteins(FIGS. 2 and 3).

[0112] So far, two nuclear transition signals (NLSA: an SV-40-like motifand NLSB: a 2-factor motif) have been identified, which are involved intransport of maize Opaque2 (O2) proteins from cytoplasm into nucleus(Varagona M. J. et al. Plant Cell 4: 1213-1227, 1992). These motifs weresearched on RISBZ1 and sequences homologous to NLSA and NLSB were foundat the same sites as O2 (101 to 135 and 232 to 264).

EXAMPLE 3 Genomic Structure of the RISBZ1 Gene

[0113] Using primers designed from the nucleotide sequence of the RISBZLcDNA, the genomic region encoding promoter and RISBZL protein wasisolated. The PCR reaction was performed using rice genomic DNA as atemplate and two pairs of oligonucleotide primers (RIS1f:5′-ATGGGTTGCGTAGCCGTAGCT-3′/SEQ ID NO: 18 and RELr5:5′-TTGCTTGGCATGAGCATCTGT-3′/SEQ ID NO: 19) and (RELf2:5′-GAGGATCAGGCCCATAT-3′/SEQ ID NO: 20 and RIS1r:5′-TCGCTATATTAAGGGAGACCA-3′/SEQ ID NO: 21). DNA fragments were amplifiedusing TAKARALA Taqpolymerase (TAKARA) in a thermal cycler through30-cycle reactions for 10 sec at 98° C., for 30 sec at 56° C. and for 5min at 68° C. The promoter region of the RISBZ1 gene was also amplifiedby thermal asymmetric interlaced (TAIL) PCR, based on the method by Liuet al, in which three oligonucleotides were used as specific primers,tail1: 5′-TGCTCCATTGCGCTCTCGGACGAG-3′/SEQ ID NO: 22, tail2:5′-ATGAATTCGCGAGGGGTTTTCGA-3′/SEQ ID NO: 23, and tail3:5′-GTTTGGGAGAAATTCGATCAAATGC-3′/SEQ ID NO: 24.

[0114] The results revealed that the RISBZ1 gene comprises of six exonsand five introns (FIG. 4). The constitution of exon/intron in thisRISBZ1 gene was identical to that of the maize O2 (Hartings H. et al.EMBO J. 8: 2795-2801, 1989), Sorghum O2 (Pirovano L. et al. Plant Mol.Biol. 24: 515-523, 1994), adlay O2 (Vettore A. L. et al. Plant Mol.Biol. 36: 249-263, 1998), and barley BLZ1 (Vicente-Carbojos J. et al.Plant J. 13: 629-640, 1998) genes (FIG. 4).

[0115] The transcription initiation site of the RISBZ1 gene wasdetermined by the primer extension analysis according to the method ofSambrook et al. (Sambrook J. et al. Molecular Cloning: A LaboratoryManual, 2nd Ed., pp. 7.79-7.83, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.). Specifically, a primer,5′-ATGGTATGGTGTTCCTAGCACAGGTGTAGC-3′ (SEQ ID NO: 25), was produced bylabelling with T4 kinase, the 5′ end of the oligonucleotide comprising30 nucleotides, which was complementary to a sequence immediatelydownstream of a desired region. Reverse transcription reaction wasconducted using this primer and 5 μg of mRNA as a template, and aSuperscript reverse transcriptase kit (Gibco BRL, Paisly, UK). Thisreaction was carried out in a mixture comprising 20 mM Tris-HCl, 50 MMMgCl₂, 10 mM DTT, 500 μM dNTP, 100,000 cpm primer, 5 μg mRNA, and200-unit Superscript reverse transcriptase (Gibco BRL, Paisly, UK), for50 min at 42° C.

[0116] As a result, the transcription initiation site was mapped to the245-nt upstream region from the translation initiation codon of theRISBZ1 gene. A ‘TATA’ box was localized at −30 to −35-nt from thetranscription initiation site. Three ‘ACGT’ motifs were found in the63-, 123-, and 198-bp upstream regions from the transcription initiationsite but none of motifs responsible for expression of seed-specificgenes, such as, GCN4 and ‘AACA’ were found. In contrast, a number of therecognition sequences for D of domain protein, ‘AAAG’, were found. Thesemotifs may be involved in stage- and/or tissue-specific expression ofthe RISBZ1 gene. For example, if the ‘ACGT’ motif is a target sequenceof the RISBZ1 protein, the RISBZ1 gene may be autoregulated by itself.However, when the RISBZ1 promoter/GUS reporter gene and the ³⁵S CaMVpromoter/RISBZ1 gene were introduced into protoplast cells, notranscriptional activation of the reporter gene was observed. These datasuggest that the RISBZ1 promoter has no target sequence for the RISBZ1protein; namely, the ‘ACGT’ motif found in the RISBZ1 promoter is not atarget sequence of the protein. Therefore, the RISBZ1 gene is probablynot autoregulated. In contrast, upon overexpression of the rice prolaminbox binding factor (RPBF) gene (which recognizes the Dof domain)transcription of the RISBZ1 promoter/GUS reporter gene is activated.This suggests that the recognition sequences of the Dof domain proteinsare involved in specific expression of the RISBZ1 gene.

EXAMPLE 4 Tissue-Specificity of the RISBZ mRNA

[0117] Northern blotting was carried out to analyze the expression ofthe RIABZ gene. According to the method by Takaiwa et al. (Varagona M.J. et al. Plant Cell 4: 1213-1227, 1992), total RNA was extracted from 5to 30 DAF seeds, roots, and seedling (5-, 10-, 15-, 20- and 30-DAF), andwas transferred to membrane filters after fractionation by agarose gelelectrophoresis. As probes, the following DNA fragments ranging from thedownstream sequence of the bZIP domain-encoding region to the 3′non-coding region in the RISBZ cDNA were used: RISBZ1, 354-bp rangingfrom 1388^(th) to 1742^(nd) nucleotides; RISBZ2, 346-bp ranging from1351^(st) to 1696^(th) nucleotides; RISBZ3, 486-bp ranging from 741^(st)to 1226^(th) nucleotides; and RISBZ5, 621-bp ranging from 742^(nd) to1362^(nd) nucleotides.

[0118] Hybridization was carried out in a solution containing 5×SSC, 5×Denhard's solution, 0.1% SDS, and 50% formamide, at 45° C. After thehybridization, the membrane filters were washed twice for 30 min with amixed solution comprising 2×SSC and 0.1% SDS, and then twice for 30 minwith a mixture comprising 0.1×SSC and 0.1% SDS.

[0119] As shown in FIG. 5, the RISBZ1 gene was expressed only in seeds,not in other tissues analyzed. The largest amount of the RISBZ1 mRNA wasaccumulated in seeds harvested from 5 DAF to 10 DAF. Such a highaccumulation of mRNA was maintained until 15 DAF, and graduallydecreased towards maturing. The peak of the RISBZ1 gene expressionappeared at an earlier stage than that of the glutelin gene. Theglutelin mRNA expression was detected from 5 DAF, had a peak at 15 DAF,and was then gradually decreased (FIG. 5). This result suggests that theRISBZ1 acts as an activator of the glutelin gene. Similar expressionpatterns have also been reported in the maize O2 (Hartings H. et al.EMBO J. 8: 2795-2801, 1989), wheat SPA (Albani D. et al. Plant Cell 9:171-184, 1997), and barley BLZ2 genes (Onate L. et al. J. Biol. Chem.274: 9175-9182, 1999).

[0120] The RISBZ2 was expressed in all the tissues analyzed. The RISBZ3and RISBZ 4 were expressed specifically in seeds at later stages ofmaturing (FIG. 5). The RISBZ3 and RISBZ 4 mRNA levels graduallyincreased until 20DAF and then decreased. The expression level of RISBZ5was extremely low, compared with other RISBZ genes, and its mRNA peakwas at 10 DAF.

EXAMPLE 5 Expression of the RISBZ1 Promoter/GUS Reporter Gene Constructin Transformants

[0121] To examine an expression pattern of the RISBZ1 gene, the sequencefragment ranging from −1674 to +213 nt numbering from the transcriptioninitiation site, was ligated upstream of GUS gene. This reporter genewas introduced into rice plant by using Agrobacterium (FIG. 6A).Transformed rice plant (Oryza sativa L. c. v. kitaake) was constructedas follows. Two oligonucleotide primers with the PstI or BamHIrestriction site at its 5′ end, 5′-AAAACTGCAGTTTTCTGA-3′ (SEQ ID NO: 26)and 5′-AATGGATCCGCGAGGGGTTTTCGAA-3′ (SEQ ID NO: 27), were used toamplify the 5′-end regions (from −1674^(th) to +4^(th) and from−1674^(th) to +213rd) of the RISBZ1 gene by PCR. The PCR reaction wascarried out in a reaction mixture (10 mM Tris-HCl pH 8.3, 1.5 mM MgCl₂,50 mM KCl, 0.01% (w/v) gelatine, 200 μM dNTPs, 1 μM primers, 0.5 μgtemplate DNA, and 2.5-unit TaqI polymerase) by 30 cycles of incubationfor 1 min at 94° C., for 1 min at 50° C. and for 2 min at 72° C. Afterdigestion with restriction enzymes, PstI and BamHI, the PCR product wascloned into the plasmid vector pBI201, and was cleaved with restrictionenzymes, PstI and SacI. The resulting DNA fragment containing the RISBZ1promoter/GUS gene was inserted between the Sse8387I and SacI sites ofthe binary vector p8cHm, which contains the CaMV35S promoter/hygromycinphosphotransferase (HPT) gene. Transformation was performed according tothe method described in Goto F. et al. Nature Biotech. 17: 282-286.

[0122] The reporter plasmid was constructed as follows. 1×21 bp, 3×21bp, and 5×21 bp of GCN4 motifs/GUS genes, as constructed by Wu et al.(Wu C. Y. et al. Plant J. 14: 673-683, 1998), were used as the reporter.A pair of 48-bp oligonucleotides with overhanged (ACGT) 5′ ends, whichwere complementary to each other, was associated to construct tetramerscomprising 12-bp wild-type GCN4 motif (GCTGAGTCATGA/SEQ ID NO: 8) andmutant GCN4 motif (GCTTCCTCATGA/SEQ ID NO: 28). These double-strandedoligonucleotide were inserted into the SalI and StuI sites of the−46CaMV/GUS reporter gene.

[0123] Transient assay for rice callus protoplast was carried outaccording to the method described by Wu et al. The GUS activity wasmeasured according to the method of Jefferson (Jefferson R. A. PlantMol. Biol. Rep. 5: 387-405, 1987), by measuring fluorescence intensityof 4-methyl-umbelliferone derived from the glucuronide precursor. UsingBio Rad Kit, the concentration of proteins was measured. Bovine serumalbumin was used as a standard protein.

[0124] As shown in FIG. 6B, high GUS activities was observed in thealeulon and sub aleulon layers of maturing seeds, but not in germs. TheGUS activity was not detected in roots, leaves, and stems even by highlysensitive fluorescence measurement. These results indicate that theRISBZ1 gene is expressed exclusively in the aleulon and sub aleulonlayers. To examine the role of the 5′-end untranslated region and uORF,the GUS activity was compared with that of a plant, which lacked uORFranging from −1674^(th) to +4^(th) numbering from the transcriptioninitiation site (FIG. 6A). As a result, no change in the expression sitewas observed due to the lack of uORF (FIG. 6B), but 5- to 10-fold weakerpromoter activities were observed (FIG. 6C). These data suggest that the5′ untranslated region may play a role in upregulation of thetranslation, in contrast to the results in the maize O2 in which uORFfunctions as a suppressor of the translation (Lohmer S. et al. PlantCell 5: 65-73).

EXAMPLE 6 Transcription Activating Ability of Five RISBZ ProteinsThrough Their Binding to the GCN4 Motif

[0125] Transcription activating ability of the five RISBZ proteinsthrough their binding to the GCN4 motif was measured by transient assay.The plasmids, into which each RISBZ1 protein-encoding sequences wereligated downstream of CaMV35S promoter as an effector, were prepared.Effector plasmids were prepared as follows. The plasmid that encodesRISBZ1 lacking its N-terminal region was prepared by PCR. In order toamplify cDNA encoding the regions ranging from 41^(st), 81^(st),121^(st), and 161^(st) amino acids numbering from the N-terminus ofRISBZ1 to its C-terminus the following primers were designed:

[0126] Forward Primers Forward primers RIS1-1:5′-AACCATGGTGCTGGAGCGGTGCCCGT-3′ (SEQ ID NO: 29) RIS1-2:5′-AACCATGGCGGCGGAGGCGGCGGCG-3′ (SEQ ID NO: 30) RIS1-3:5′-CCCCATGGAGTACAACGCGATGC-3′ (SEQ ID NO: 31) RIS1-4:5′-AACCATGGTTGGTTCCATCCTGAGT-3′ (SEQ ID NO: 32) RIS1-5:5′-AACCATGGCTCATGCCAAGCAAGCT-3′ (SEQ ID NO: 33) RIS1-6:5′-AACCATGGATGAAGAAGATAAAGTGAAG-3′ (SEQ ID NO: 34) Reverse primerBRIS1R: 5′-TAGGATCCGCTCCTACTACTGAAGCT-3′. (SEQ ID NO: 35)

[0127] BRIS1R: 5′-TAGGATCCGCTCCTACTACTGAAGCT-3′ (SEQ ID NO: 35).

[0128] These primers were designed to have an NcoI or BamHI restrictionsite at their 5′ end. Since a translational initiation codon was lost bydeletion of its N-terminal region, ATG of the NcoI restriction site wasutilized. cDNAs were amplified by PCR comprising incubation for 2 min at94° C., 30-cycle reaction for 1 min at 94° C., for 1 min at 50° C., andfor 2 min at 72° C., followed by incubation for 5 min at 72° C. The PCRproducts were digested with restriction enzymes, NcoI and BamHI, andthen purified through agarose gel electrophoresis. The purified cDNAfragments were finally inserted into the pRT100 vector (Topfer R. et al.Nucl. Acids Res. 15: 5890, 1987).

[0129] Plasmids encoding the fusion proteins comprising GAL4 DNA-bindingdomain (amino acid residues from 1^(st) to 147^(th)) and the RISBZ1 orRISBZ2 gene were also constructed. In order to amplify the cDNA regionencoding various N-terminal region of RISBZ1 and RISBZ2 by PCR using PfuTaq polymerase (STRATAGENE), the following reverse primers, to which aBamHI site, a terminal codon, and an SstI site were added at its 5′-end,were prepared as well as the following forward primers:

[0130] Forward Primers Forward primers RISBZ1-F1:5′-AAGGATCCAATGGAGCACGTGTTCGCC-3′ (SEQ ID NO: 36) RISBZ1-F2:5′-AAGGATCCGGCGGCGGAGGCGGCGCG-3′ (SEQ ID NO: 37) RISBZ1-F3:5′-GCCGGATCCAGTTGGTTCCATCCTGAG-3′ (SEQ ID NO: 38) RISBZ1-F4:5′-AAGGATCCTGATGAAGAAGATAAAGT-3′ (SEQ ID NO: 39) RISBZ1-F1-2:5′-AAGGATCCAGGAGTAGATGACGTCGGC-3′ (SEQ ID NO: 40) RISBZ1-F1-3:5′-AAGGATCCAGACGAGATCCCCGACCCGCT-3′ (SEQ ID NO: 41) Reverse primersRISBZ1-R1: 5′-TAGAGCTCTACGCCGCCGGCATCGGGCT-3′ (SEQ ID NO: 42) RISBZ1-R2:5′-TAGAGCTCTAAAGGATCATATTTCCCAT-3′ (SEQ ID NO: 43) RISBZ1-R1-1:5′-TAGAGCTCTAGGCGGCCGCCGCCGGCTG-3′ (SEQ ID NO: 44) RISBZ1-R1-2:5′-TAGAGCTCTACGGCGGCGGCGGAGCCCA-3′. (SEQ ID NO: 45)

[0131] cDNAs encoding various N-terminal regions of RISBZL and RISBZ2were amplified by PCR comprising incubation for 2 min at 94° C., 30cycles of reaction for 1 min at 94° C., for 1 min at 50° C., and for 1min at 72° C., and then incubation for 5 min at 72° C., using theabove-described primers. The amplified cDNAs were digested with BamHIand SacI restriction enzymes, and were purified by 2% agarose gelelectrophoresis. The purified cDNA fragments were ligated downstream ofthe GAL4 DNA domain-encoding region in the ³⁵S-564 vector digested withthe same restriction enzymes so that their reading frames were matched.Mutations were also introduced into the N-terminal regions of RISBZ1 byPCR mutagenesis. The cDNA sequences were confirmed, and their partialsequence from 1^(st) to 57^(th) amino acid residues was amplified byPCR. The products were ligated downstream of the GAL4 DNAdomain-encoding region in their reading frames.

[0132] In addition, reporter plasmids, into which the GUS gene, and oneor three repeat(s) of the 12-bp GCN4 motif or one or five repeat(s) ofthe 21-bp GCN4 motif were inserted, were constructed. For negativecontrol experiments, a reporter plasmid comprising four repeats of amutant 12-bp GCN4 motif and the GUS reporter gene was used. The mutant12-bp GCN4 motif has a mutation in the target sequence that isrecognized by the RISBZ1 and O2. These plasmid constructs wereintroduced alone or in combination with other reporter or effectorplasmid into rice protoplast cells prepared from its callus culture, andthe GUS activity was assayed. When the reporter plasmid or effectorplasmid was introduced alone into the protoplast, the GUS activity wasdetected at a low level. As shown in Table 1, however, in the presenceof ³⁵S/RISBZ1 or ³⁵S/O2, which were introduced as effector plasmids, thetranscription of the reporter gene was activated. Even in the presenceof these effector plasmids, the transcriptional activity of the GUS genedownstream of the mutant 12-bp GCN4 motif was the same level as that ofbackground. These results indicate that the RISBZ1 gene productactivates the reporter gene mediated by the GCN4 motif. Thetranscriptional activity of the reporter gene induced by the RISBZ1 geneproduct was slightly higher than that induced by the O2 gene product. Asshown in Table 2, the activity induced by RISBZ1 was enhanced dependingon the copy number of the GCN4 motif. 1 to 12 copies of 21-bp GCN motifwere assayed, and the transcriptional activity was enhancedproportionately up to 9 copies. However, even though the other RISBZgenes were expressed under the control of the ³⁵S CaMV promoter, thetranscriptional activity of the reporter gene was less than or equal to1.4% of that induced by the RISBZ1 or O2 gene product. Thus, it wasrevealed that only the RISBZ1 protein can activate the transcriptionthrough its binding to the GCN4 motif. TABLE 1 Effector GUS activity (pM4-MU/min/mg protein) 35S/Opaque2 2658 ± 318 35S/RISBZ1 2994 ± 15735S/RISBZ2 44 ± 7 35S/RISBZ3  1.3 ± 1.2 35S/RISBZ4 17.3 ± 0.9 35S/RISBZ5  31 ± 8.8

[0133] The 4×12-bp GCN4 motifs/GUS reporter gene was introduced intoprotoplast cells together with the effector plasmid, and the GUSactivity was measured. Data were obtained from three independentmeasurements. TABLE 2 Effector GUS Activity (pM 4-MU/min/mg protein)Reporter (−) (+) RISBZ1 (+) Opaque2 1 × 12-bp GCN4 32 ± 1.5 295 ± 4.5 182 ± 6  (9.2*) (5.6*) 4 × 12-bp GCN4 21  604 ± 24.5 452 ± 7.5  (28.7*)(21.5*) 1 × 21-bp GCN4 30 ± 3   1318 ± 55.5  1139 ± 22.5  (43.9*)(37.9*) 5 × 21-bp GCN4 104 13222 ± 1094  11932 ± 22.5   (127.1*)(114.7*)

[0134] As a reporter, the 1×12-bp, 4×12-bp, 1×21-bp or 5×21-bp GCN4motif/GUS gene was used. This table shows the GUS activity induced bythe expression of RISBZ1 (+RISBZ1) gene or by Opaque2 (+Opaque2) gene.

EXAMPLE 7 Binding Site of the RISBZ1 Protein

[0135] The present inventors have previously discovered that the O2protein recognizes the GCN4 motif (TGAGTCA) that is present in thepromoter region ranging from −165^(th) to −160^(th) of GluB-1, aglutelin gene (Wu C. Y. et al. Plant J. 14: 673-683, 1998). By amethylation interference experiment, the present inventors have alsodetermined the binding site of the RISBZ1 protein in the promoter regionof the GluB-1 gene.

[0136] Production and purification of the GST-RISBZ1 fusion protein wereperformed as follows. Five coding regions from RISBZ1 cDNA wereamplified by PCR using oligonucleotide primers to which the followingappropriate restriction enzyme sites were added at their 5′ end;BamHI-blunt ends for RISBZ1, BamHI-XhoI for RISBZ2, BamHI-SalI forRISBZ3, BamHI-SalI for RISBZ4, and BamHI-XhoI for RISBZ5. Afterdigestion with the restriction enzymes, the PCR products were ligatedinto the cloning sites of the pGEX-4T-3 vector (Amersham PharmaciaBiotech). The GST-RISBZ fusion protein was expressed according to themethod of Suzuki et al. (Suzuki A. et al. Plant Cell Physiol. 39:555-559, 1998). After affinity purification, the GST fusion protein wasdialyzed against a binding buffer comprising 20 mM HEPES-KOH pH 7.9, 50mM KCl, 1 mM EDTA, and 10% glycerol, for four hours, and immediatelystored at −80° C.

[0137] Methylation interference experiment was performed as described byWeinberger et al. (Weinberger J. et al. Nature 322: 846-849, 1986). The5′-flanking region (from −245^(th) to +18th nucleotides) of the GluB1gene was digested with restriction enzymes, SalI and BamHI, and the endsof the fragment was labeled with [α-³²P] dCTP by a ‘fill-in’ reaction.The labelled fragment was methylated by treating it with dimethylsulphate, mixed with GST-RISBZ1, and then incubated. Usingnon-denaturing acrylamide gel (5%, 0.25×TBE) electrophoresis, the DNAfragment complexed with GST-RISBZ1 and free DNA fragments were separatedfrom each other. These DNA fragments were further purified by DEAESepharose column chromatography, were treated with piperidine, and werefractionated by 6% denaturing acrylamide gel electrophoresis.

[0138] As shown in FIG. 7, the GST-RISBZ1 fusion protein protectedguanine residues that locate in the −165^(th) to −160^(th) region of theGluB-1 promoter. The guanine residues protected were the same residuesprotected in the O2 promoter (Albani D. et al. Plant Cell 9: 171-184,1997). A guanine residue present in the ‘ACGT’ motif (also termed as anA/G hybrid box) at the ˜79^(th) to −76^(th) residues in the promoterregion ranging from −197^(th) to +18th, was not protected.

[0139] Furthermore, gel shift assay was conducted as described below toexamine whether the RISBZ1 protein can recognize the GCN4 motif.

[0140] A pair of oligonucleotides complementary to each other, which wasprepared by adding TCGA sequence was added to 21-nt fragment of GluB1promoter region (from −175^(th) to −155^(th)), was labeled at its endswith [α-³²P] dCTP by ‘fill-in’ reaction for use as a probe. Seven pairsof complementary oligonucleotides with mutations every three contiguousnucleotides (FIG. 8A) were also synthesized for use as mutant competitorfragments and were annealed. Gel shift analysis using the GST fusionprotein was carried out by a method described by Wu et al. (Wu C. Y. etal. Plant J. 14: 673-683, 1998) and by Suzuki et al. (Suzuki A. et al.Plant Cell Physiol. 39: 555-559, 1998). The labeled oligonucleotideprobe was mixed with 0.5 μg of the GST-RISBZ fusion protein, andincubated for 20 min at room temperature. In competition experiments,the competitor fragment was added to the mixture at the 100-fold orhigher molecular weight ratio. The reacted mixture was analyzed bynon-denaturing acrylamide gel (5%, 0.25×TBE) electrophoresis.

[0141] The detection of shift bands showed that the GST-RISBZ1 proteinwas able to bind to the 21-bp DNA fragment containing the GCN4 motif(FIG. 8B). Furthermore, as shown FIG. 8A, the 21-bp DNA fragments withmutation in every three contiguous nucleotides were used as competitorsand examined. When the DNA fragments with the mutations in the GCF motifwere added as the competitor, the binding of the DNA fragments that wereadded as probes was hardly or not inhibited at all (FIGS. 8B to F). Bycontrast, when the DNA fragments with mutations in the franking sequenceof the GCN4 motif were added as the competitor, the shift bandsdisappeared (FIGS. 8B to F). Since the mutation of the GCN4 motifmarkedly affects the binding of the RISBZ1 protein to the motif, it wasrevealed that the RISBZ1 protein recognizes the GCN4 motif sequencespecifically. The similar experiments carried out using the other RISBZproteins revealed that all the RISBZ proteins could specificallyrecognize the GCN4 motif. As shown in FIGS. 8B to F, the affinity ofeach RISBZ proteins for the GCN4 motif slightly varies. In the cases ofRISBZ2 and RISBZ5, when the DNA fragments with mutations in the frankingsequence of the GCN motif were used as the competitor, the shift bandswere not disappeared completely (FIGS. 8C and F).

[0142] From these results, it was revealed that the RISBZ proteinsspecifically recognize the GCN4 motif with slightly variable affinities.

EXAMPLE 8 Ability of RISBZ1 Protein to Form a Heterodimer

[0143] It was considered that the RISBZ1 protein, a bZIP-typetranscription factor, binds to the GCN4-like motif upon forming aheterodimer with other RISBZ proteins. Therefore, the ability of RISBZ1to heterodimerize with RISBZ2 or RISBZ3 was examined. The full-lengthRISBZ1 protein, and short-form-RISBZ2 protein (sRISBZ2) and short-formRISBZ3 protein (sRISBZ3) were prepared using wheat germ extracts (FIG.9A), and were used for DNA binding assay. The in vitro translation wascarried out as follows. The coding region of RISBZ1 cDNA and the bZIPdomain-encoding regions of RISBZ2 cDNA and RISBZ3 cDNA were amplifiedusing the following forward primers with the NcoI site at their 5′ endsand reverse primers encoding a terminator codon and the BamHI site; ForRISBZ1 For RISBZ1 R1F: 5′-AAACCATGGAGCACGTGTTCGCCGT-3′ and (SEQ ID NO:46) BRIS1r: 5′-TAGGATCCGCTCCTACTACTGAAGCT-3′; (SEQ ID NO: 47) ForsRISBZ2 dR2-1: 5′-AAACCATGGAGGGAGAAGCTGAGACC-3′ and (SEQ ID NO: 48)R2ra1: 5′-AAAGGATCCTACATATCAGAAGCGGCGGGA-3′; and (SEQ ID NO: 49) ForsRISBZ3, dR3-1: 5′-AAACCATGGATATAGAGGGCGGTCCA-3′ and (SEQ ID NO: 50)R3ral: 5′-AAAGGATCCTACAGCCCGCCCAGGTGGCCG-3′. (SEQ ID NO: 51)

[0144] PCR amplification was carried out in a reaction mixturecomprising 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl₂, 50 mM KCl, 0.01% (w/v)gelatine, 200 μM dNTPs, 1 μM primers, 0.5 mg template DNA, and 2.5-unitTaqI polymerase by 30 cycles of incubation for 1 min at 94° C., for 1min at 50° C. and for 2 min at 72° C.

[0145] The PCR products were digested with restriction enzymes, NcoI andBamHI, and were ligated into the pET8c cloning vector (Novagen) toconstruct plasmids. Using these plasmids as templates, in vitrotranscription/translation (TNT coupled wheat germ extract systems;Promega) was performed for the production of the full-length RISBZ1protein, and short-form-RISBZ2 (RISBZ2s) and -RISBZ3 (RISBZ3s). For gelshift assay, 4 μl of the wheat germ extract that was used in the abovereaction was used.

[0146] Gel shift assay was employed to separate homodimers andheterodimers bound to the 21-bp GCN4 motifs. After pre-incubating RISBZ1with sRISBZ2 and sRISBZ3, the DNA probes comprising the GCN4 motif wereadded to the incubation mixture. The results indicate that RISBZhomodimers as well as heterodimers can bind to the GCN4 motif.Therefore, it was demonstrated that the RISBZ proteins form heterodimerswith the other members of the RISBZ family.

EXAMPLE 9 Involvement of the N-Terminal Region of the RISBZ1 Proteins inthe Transcriptional Activation

[0147] Transient assay was performed to identify the domain of theRISBZ1 protein involved in transcription activation. The GUS gene, towhich three copies of the 21-bp GCN4 motif and the core promotersequence of CaMV35S were connected, was prepared as a reporter. Variousdomains of the RISBZ1 proteins were expressed using the CaMV35S promoterin order to examine if these domains can activate the reporter gene.

[0148] A series of effector plasmids encoding RISBZ1 proteins in whichevery 40 amino acids from N-terminus to the basic domain were deleted(encoding the amino acids region ranging from 41^(st) to 436^(th),81^(st) to ₄₃₆, 121^(st) to 436^(th), 161^(st) to 436^(th), 201^(st) to436^(th), or 235^(th) to 436^(th) in the amino acid sequence set forthin SEQ ID NO: 2), were constructed. When the effector plasmid encodingthe full-length RISBZ1 protein and the reporter plasmid (the GUS gene towhich four copies of the 12-bp GCN motif and the core promoter sequenceof CaMV35S were linked) were introduced into protoplasts, approximately30-fold higher activity of GUS was detected compared to that ofprotoplast into which the reporter plasmid alone was introduced. Whenthe transcriptional activity of this reporter gene was set as 100%, theactivity of the gene with deletion of the first 40-amino acid wasdecreased to 20%. Furthermore, the activity of the reporter gene wasdecreased gradually to 10% by deleting each 40 amino acids. Hence, itwas suggested that the N-terminal 40 amino acid residues of RISBZ1 aremainly involved in the transcription activation.

[0149] To further analyze the association of the N-terminal 40 aminoacids of RISBZ1 with its transcription activating ability, variousfusion proteins between the DNA binding domain of the yeasttranscriptional activating factor GAL4 and various portions of theRISBZ1 protein were constructed and expressed for the gain-of-functionassay. As shown in FIG. 10, a plasmid, in which the coding sequences offused proteins comprising the GAL4-DNA binding domain and variousregions of RISBZ1 were connected downstream of the CaMV35S promoter, wasconstructed and used as an effector. These effector plasmids wereintroduced into protoplast together with a reporter construct (the GUSgene, to which nine copies of the GAL4-DNA binding site and CaMV35S corepromoter were connected).

[0150] The significant difference was not found in transcriptionactivating ability of the fusion protein comprising the GAL4-DNA bindingdomain and the partial amino acid sequence from 1st to 235^(th) aminoacids of RISBZ1, compared with that of a series of the fused proteins inwhich amino acids were deleted towards the 27^(th) residue from theC-terminal residue of RISBZ1 (FIG. 10). The transcription activatingability of the fusion protein with the first 20 amino acid residues weredramatically decreased (FIG. 10). A fusion protein with deletion of theN-terminal eight residues of RISBZ1 lost the transcriptional activity.In contrast, fusion proteins comprising the GAL4-DNA binding domain andother region of RISBZ1 (from 27^(th) through 57^(th), 81^(st) through234^(th), 161^(st) through 234^(th), or 235^(th) through 436^(th) in SEQID NO: 2) had no effect on the transcriptional activity of the reportergen. These results suggest that the proline-rich domain within theN-terminal 27 amino acid residues of the RISBZ1 protein, rather than theacidic domain, involves in the transcription activation.

EXAMPLE 10 Difference Between RISBZ1 and Other RISBZ Proteins inTranscription Activating Ability Analyzed by Domain Swapping

[0151] Although all the members of the RISBZ protein family have similaraffinity for the GCN4 motif sequence, only the RISBZ1 has thetranscription activating ability. To find out the reason of thisdifference, domain swapping between RISBZ1-, and RISBZ2- orRISBZ3-protein was carried out. The N-terminal region at 1^(st) through299th of RISBZ1, which resides upstream of the bZIP domain, was replacedwith the N-terminal region, 1^(st) through 229^(th) of RISBZ2 or 1^(st)through 137^(th) of RISBZ3.

[0152] Fusion proteins that have the N-terminal region of RISBZ1together with the DNA binding domain of RISBZ2 or RISBZ3 showed onlyapproximately 15% or 38% of the transcription activating ability,respectively, compared with that of the full-length RISBZ1. In contrast,fusion proteins that have the N-terminal region of RISBZ2 or RISBZ3together with the RISBZ1 DNA binding domain showed a slightly highertranscription activity than that induced by the RISBZ1 DNA bindingdomain alone.

[0153] These results indicate that the N-terminal region is mainlyinvolved in the transcription activation. The lower level of the RISBZ2or RISBZ3 transcription activating ability may be due to deletion ormutation of the region corresponding to RISBZ1 transcription activatingdomain during evolution. Alternatively, the formation process oftranscription activating domain may be responsible for that. It ishighly possible that the lower activity of RISBZ3 is due to the lack ofthe proline-rich domain present in RISBZ1. This applies to RISBZ4 andRISBZ5. The results of the gel shift assay probably exclude thepossibility that the differences of affinity with GCN4 motif raise thedifferences of transcription activating ability.

[0154] The proline-rich domain of RISBZ1 was also highly conserved inRISBZ2, but the transcription activating ability of RISBZ2 was extremelylow compared to that of RISBZ1. When an effector plasmid that encodes afused protein comprising the N-terminal 27 amino acid residues of RISBZ2including proline-rich domain and the GAL4-DNA binding domain wasintroduced together with a reporter plasmid encoding the GCN4 motifconnected to the GUS gene into protoplast, no increased activity of GUSwas observed.

[0155] Since only eight-residue differences among the N-terminal 27residues were observed between RISBZ1 and RISBZ2, the present inventorshave examined which of the residues among the eight are responsible forthe difference in transcription activating ability. The eight amino acidresidues of RISBZ1, which were different from RISBZ2, were replaced oneby one with the residues of RISBZ2, and the resulting chimericN-terminal sequences comprising 40 amino acids were fused with theGAL4-DNA binding domain to construct effector plasmids encoding thefused proteins. These effector plasmids were introduced into protoplasttogether with the reporter plasmid in which the GCN4 motif was fusedwith the GUS gene. Among eight effector plasmids, all the effectorconstructs, except for those encoding a protein with replacement of theseventh residue counting from the N-terminus of RISBZ1, did not activatethe transcription of the reporter gene. It was presumed using the Kyteand Doolittle formula that all these seven substitutions of amino acids,which were lost transcription activating ability, would induce thechange of a hydropacy pattern (FIG. 11).

EXAMPLE 11 Use of the Transcription Factor RISBZ1 for Plant Breeding

[0156] The present inventors have examined the possibility to use thetranscription factor, RISBZ1, which has a transcription activatingability for plant breeding. In order to specifically overexpress thetranscription factor in seeds, rice-plants were transformed with aplasmid construct that encodes the RISBZ1 gene under the control of thepromoter of the rice prolamin gene, which encodes a seed storageprotein, with 13-kDa molecular masses. The DNA fragment ranging from theEcoRI site, located at the −29^(th) position, to the poly (A) additionsite of the RISBZ1 gene was linked to the prolamin promoter encompassingfrom the −652^(nd) through −13^(th) from the translation initiation siteATG of the gene. The construct was inserted into the binary pGTV-Barvector, and the resulting vector was introduced into rice plants usingAgrobacterium. By this approach, 28 independent transformed lines wereestablished. Screening of rice plants that overexpress the RISBZ1 mRNAwas carried out by Northern hybridization of RNA extracted from maturingseeds using cDNA of RISBZ1 as a probe (FIG. 12). These linesoverexpressing RISBZ1 were crossed with the transformed rice plants, inwhich a plasmid construct encoding five tandem repeats of the 21-bp GCN4motif (5′-GTTTGTCATGGCTGAGTCATG-3′/SEQ ID NO: 52), a target of theRISBZ1 protein, linked to the minimum promoter/GUS reporter had beenintroduced.

[0157] As a result, it was revealed that the expression level of GUSreporter genes were, due to overexpression of RISBZ1 enhanced by400-times or more (450 to 750 nmol/min/mg protein) than that ofcontrols, 5×GCN4 lines (11 to 14 nmol/min/mg protein) (FIG. 12). Theseresults suggest that the transcription of foreign genes can be highlyactivated by connecting the foreign genes downstream of the targetsequence of the transcription factor RISBZ1 with transcriptionactivating ability and overexpressing RISBZ1.

[0158] The RISBZ1 proteins can activate not only the glutelin gene butalso other storage protein genes. The ³⁵S CaMV promoter/RISBZ1 fusionconstruct together with the glutelin promoter/GUS, glutelin promoter(−980^(th) to ATG)/GUS, or 13-Kd prolamin promoter (from −652^(nd) to−29th)/GUS, was introduced into rice protoplast using electroporation,and the transient expressions of them were examined.

[0159] The results indicated that the RISBZ1 protein bound to the targetsequences containing GCN4 motifs in these promoters and activated thetranscription of the foreign genes. It was revealed that thetranscriptions were activated 5 to 10-fold in the case of the 13-Kdprolamin promoter and 20 to 30-fold in the case of the globulinpromoter, higher than that of the background. Therefore, methylationinterference reaction was used to determine how RISBZ1 recognizes thenucleotide sequences of these genes.

[0160] The results showed that three GCN4 motifs (TGACACA, GATGACTCA,and TGACTCAC) of the prolamin gene and three motifs different from theGCN4 motif (GGTGACAC, GTATGTGGC, and GATCCATGTCAC) of the globulin genewere recognized by the RISBZ1 protein. To determine specific sequencesin the promoters that are recognized by the RISBZ1, transient expressionof the GUS gene was examined by using a chimeric promoter sequence inwhich the G, A, C, G/C, A/G, or C/A box, GCN4, 22-Kd zein binding siteand four repeats of 12-bp sequence including the b-32 binding site wereinserted in tandem into the −46 CaMV ³⁵S core promoter/GUS reportergene. The results indicate that the RISBZ1 protein preferentiallyrecognizes the G/C box and GCN4 motif (FIG. 13).

[0161] Furthermore, it was studied to see if the RISBZ1 proteinrecognized various distinct GCN4 motifs present in the promoter for thestorage protein genes. The results indicate that the flanking sequencesof the core sequence ‘TGAGTCA’ of GCN4 motif influence transcriptionactivating ability, and that the GCN4 motifs of the wheat gliadin geneand rye secalin gene have high transcription activating ability (FIG.14).

INDUSTRIAL APPLICABILITY

[0162] The present invention provides novel transcription factors thatregulate the expression of rice seed storage proteins, and genes thatencode the transcription factors. It is expected that the expression ofmany seed storage proteins regulated by the RISBZ1 protein of thepresent invention having transcription activating ability can beenhanced by introducing the gene encoding the RISBZ1 protein into cellsto overexpress it. The present invention also provides novel geneexpression systems in which a useful foreign gene, encoding such as anantibody and an enzyme, can be highly expressed using the transcriptionfactor of the present invention, by linking the recognition sequence ofthe transcription factor, the GCN4 motif, in tandem and introducing itinto the promoter for a gene encoding a seed storage protein tofacilitate its binding to the transcription factor. Thus, expression ofthe gene encoding storage protein and the useful foreign gene can begreatly enhanced under the control of the modified promoter. Thisenables abundant accumulation of a seed storage protein in endosperm,and more nutritious seeds (e.g. rice) and production of seeds in whichuseful proteins are highly accumulated.

1 52 1 1751 DNA Oryza sativa CDS (211)..(1518) 1 ggcacgagaa aaaacccatgggttgcgtag ccgtagcttt cccaccattt ccttctctcc 60 gaagcctcct cctctccgcttcctcccgcg aaaccaaatt ccaaagcatt tgatcgaatt 120 tctcccaaac ttttccagcgttttcaattt cgccccgatt tcggttcgaa aacccctcgc 180 gaattcattt caaactcgtccgagagcgca atg gag cac gtg ttc gcc gtc gac 234 Met Glu His Val Phe AlaVal Asp 1 5 gag atc ccc gac ccg ctg tgg gct ccg ccg ccg ccg gtg cag ccggcg 282 Glu Ile Pro Asp Pro Leu Trp Ala Pro Pro Pro Pro Val Gln Pro Ala10 15 20 gcg gcc gcc gga gta gat gac gtc ggc gcg gtg agc ggc ggc ggg ttg330 Ala Ala Ala Gly Val Asp Asp Val Gly Ala Val Ser Gly Gly Gly Leu 2530 35 40 ctg gag cgg tgc ccg tcg ggg tgg aac ctc gag agg ttt ctg gag gag378 Leu Glu Arg Cys Pro Ser Gly Trp Asn Leu Glu Arg Phe Leu Glu Glu 4550 55 ctc gac ggc gtc cct gca ccg gcg gcg agc ccg gac ggc gcg gcg att426 Leu Asp Gly Val Pro Ala Pro Ala Ala Ser Pro Asp Gly Ala Ala Ile 6065 70 tac cct agc ccg atg ccg gcg gcg gcg gcg gag gcg gcg gcg cgc tgg474 Tyr Pro Ser Pro Met Pro Ala Ala Ala Ala Glu Ala Ala Ala Arg Trp 7580 85 agt agg ggc tac ggc gat cgt gag gcg gtg ggg gtg atg ccc atg ccc522 Ser Arg Gly Tyr Gly Asp Arg Glu Ala Val Gly Val Met Pro Met Pro 9095 100 gcg gcc gcg ctt ccg gcg gcg ccg gcg agc gcg gcg atg gac ccc gtg570 Ala Ala Ala Leu Pro Ala Ala Pro Ala Ser Ala Ala Met Asp Pro Val 105110 115 120 gag tac aac gcg atg ctg aag cgg aag ctg gac gag gac ctc gccacc 618 Glu Tyr Asn Ala Met Leu Lys Arg Lys Leu Asp Glu Asp Leu Ala Thr125 130 135 gtc gcc atg tgg agg gcc tct ggt gca ata cat tct gag agt cctcta 666 Val Ala Met Trp Arg Ala Ser Gly Ala Ile His Ser Glu Ser Pro Leu140 145 150 ggc aat aaa aca tca ctg agt ata gtt ggt tcc atc ctg agt tcacag 714 Gly Asn Lys Thr Ser Leu Ser Ile Val Gly Ser Ile Leu Ser Ser Gln155 160 165 aag tgc att gaa ggt aac ggg ata cta gtg cag acc aag tta agtcct 762 Lys Cys Ile Glu Gly Asn Gly Ile Leu Val Gln Thr Lys Leu Ser Pro170 175 180 ggc cca aat gga gga tca ggc cca tat gta aat caa aat aca gatgct 810 Gly Pro Asn Gly Gly Ser Gly Pro Tyr Val Asn Gln Asn Thr Asp Ala185 190 195 200 cat gcc aag caa gct acg agt ggt tcc tca agg gag cca tcacca tca 858 His Ala Lys Gln Ala Thr Ser Gly Ser Ser Arg Glu Pro Ser ProSer 205 210 215 gag gat gat gat atg gaa gga gat gca gag gca atg gga aatatg atc 906 Glu Asp Asp Asp Met Glu Gly Asp Ala Glu Ala Met Gly Asn MetIle 220 225 230 ctt gat gaa gaa gat aaa gtg aag aaa agg aag gaa tcc aaccgg gag 954 Leu Asp Glu Glu Asp Lys Val Lys Lys Arg Lys Glu Ser Asn ArgGlu 235 240 245 tca gct aga cgc tca aga agc aga aag gca gct cgc cta aaagac ctg 1002 Ser Ala Arg Arg Ser Arg Ser Arg Lys Ala Ala Arg Leu Lys AspLeu 250 255 260 gag gag cag gta tca cta tta agg gtt gaa aac tct tct ctgttg agg 1050 Glu Glu Gln Val Ser Leu Leu Arg Val Glu Asn Ser Ser Leu LeuArg 265 270 275 280 cgt ctt gct gat gca aat cag aag tac agt gct gct gctatt gac aat 1098 Arg Leu Ala Asp Ala Asn Gln Lys Tyr Ser Ala Ala Ala IleAsp Asn 285 290 295 agg gta cta atg gca gac att gaa gcc cta aga gca aaggtg agg atg 1146 Arg Val Leu Met Ala Asp Ile Glu Ala Leu Arg Ala Lys ValArg Met 300 305 310 gca gag gag agt gtg aag atg gtt aca ggg gct aga caactt cac cag 1194 Ala Glu Glu Ser Val Lys Met Val Thr Gly Ala Arg Gln LeuHis Gln 315 320 325 gcc att cct gac atg caa tct ccc ctc aat gtc aac tctgat gct tct 1242 Ala Ile Pro Asp Met Gln Ser Pro Leu Asn Val Asn Ser AspAla Ser 330 335 340 gtg ccg atc cag aac aac aac cca atg aac tac ttc tccaac gct aac 1290 Val Pro Ile Gln Asn Asn Asn Pro Met Asn Tyr Phe Ser AsnAla Asn 345 350 355 360 aat gcc ggt gtt aac agc ttc atg cac cag gtt tctcca gcg ttc cag 1338 Asn Ala Gly Val Asn Ser Phe Met His Gln Val Ser ProAla Phe Gln 365 370 375 att gtg gat tct gtc gag aag att gac cca aca gatcca gtg cag ctg 1386 Ile Val Asp Ser Val Glu Lys Ile Asp Pro Thr Asp ProVal Gln Leu 380 385 390 cag cag caa cag atg gcg agc ttg cag cat ctt cagaat aga gct tgt 1434 Gln Gln Gln Gln Met Ala Ser Leu Gln His Leu Gln AsnArg Ala Cys 395 400 405 ggt ggc ggc gca agt tcg aat gaa tat aca gca tgggga tcg tct ctg 1482 Gly Gly Gly Ala Ser Ser Asn Glu Tyr Thr Ala Trp GlySer Ser Leu 410 415 420 atg gat gca aat gag ctt gtc aac atg gag ctt cagtagtaggagc 1528 Met Asp Ala Asn Glu Leu Val Asn Met Glu Leu Gln 425 430435 atatcctaac aacatgatga gagcatttgg aggtgcaaat ttgcaacctg caaatgctgt1588 tttgtagtag tagttgttgt cgctgttttt gtctgaaact gtagtttcta tggattttgg1648 acttgctgag gaacatctgc ggctgttgtt gtttcaaatt gagaaaatga gggacaatgg1708 gacatggtgg tctcccttaa tatagcgaaa aaatggttgg ata 1751 2 436 PRTOryza sativa 2 Met Glu His Val Phe Ala Val Asp Glu Ile Pro Asp Pro LeuTrp Ala 1 5 10 15 Pro Pro Pro Pro Val Gln Pro Ala Ala Ala Ala Gly ValAsp Asp Val 20 25 30 Gly Ala Val Ser Gly Gly Gly Leu Leu Glu Arg Cys ProSer Gly Trp 35 40 45 Asn Leu Glu Arg Phe Leu Glu Glu Leu Asp Gly Val ProAla Pro Ala 50 55 60 Ala Ser Pro Asp Gly Ala Ala Ile Tyr Pro Ser Pro MetPro Ala Ala 65 70 75 80 Ala Ala Glu Ala Ala Ala Arg Trp Ser Arg Gly TyrGly Asp Arg Glu 85 90 95 Ala Val Gly Val Met Pro Met Pro Ala Ala Ala LeuPro Ala Ala Pro 100 105 110 Ala Ser Ala Ala Met Asp Pro Val Glu Tyr AsnAla Met Leu Lys Arg 115 120 125 Lys Leu Asp Glu Asp Leu Ala Thr Val AlaMet Trp Arg Ala Ser Gly 130 135 140 Ala Ile His Ser Glu Ser Pro Leu GlyAsn Lys Thr Ser Leu Ser Ile 145 150 155 160 Val Gly Ser Ile Leu Ser SerGln Lys Cys Ile Glu Gly Asn Gly Ile 165 170 175 Leu Val Gln Thr Lys LeuSer Pro Gly Pro Asn Gly Gly Ser Gly Pro 180 185 190 Tyr Val Asn Gln AsnThr Asp Ala His Ala Lys Gln Ala Thr Ser Gly 195 200 205 Ser Ser Arg GluPro Ser Pro Ser Glu Asp Asp Asp Met Glu Gly Asp 210 215 220 Ala Glu AlaMet Gly Asn Met Ile Leu Asp Glu Glu Asp Lys Val Lys 225 230 235 240 LysArg Lys Glu Ser Asn Arg Glu Ser Ala Arg Arg Ser Arg Ser Arg 245 250 255Lys Ala Ala Arg Leu Lys Asp Leu Glu Glu Gln Val Ser Leu Leu Arg 260 265270 Val Glu Asn Ser Ser Leu Leu Arg Arg Leu Ala Asp Ala Asn Gln Lys 275280 285 Tyr Ser Ala Ala Ala Ile Asp Asn Arg Val Leu Met Ala Asp Ile Glu290 295 300 Ala Leu Arg Ala Lys Val Arg Met Ala Glu Glu Ser Val Lys MetVal 305 310 315 320 Thr Gly Ala Arg Gln Leu His Gln Ala Ile Pro Asp MetGln Ser Pro 325 330 335 Leu Asn Val Asn Ser Asp Ala Ser Val Pro Ile GlnAsn Asn Asn Pro 340 345 350 Met Asn Tyr Phe Ser Asn Ala Asn Asn Ala GlyVal Asn Ser Phe Met 355 360 365 His Gln Val Ser Pro Ala Phe Gln Ile ValAsp Ser Val Glu Lys Ile 370 375 380 Asp Pro Thr Asp Pro Val Gln Leu GlnGln Gln Gln Met Ala Ser Leu 385 390 395 400 Gln His Leu Gln Asn Arg AlaCys Gly Gly Gly Ala Ser Ser Asn Glu 405 410 415 Tyr Thr Ala Trp Gly SerSer Leu Met Asp Ala Asn Glu Leu Val Asn 420 425 430 Met Glu Leu Gln 4353 6335 DNA Oryza sativa 3 tgctccattg cgctctcgga cgagcatata tgtatgacatgtgggcccgg aatgtcagta 60 acagggaatc ctgaaaaaaa tgcagctatg tgataattttcaacgctaat gttgcagttt 120 tctgaaaagt gtcgcaaagg ttgcagcaaa gtgatagtttagtcaataaa actgcagttt 180 tctgaaattt actctagttt ttttacctat ctagttggttttatgtgtaa ctaaacctta 240 catcagatca aaacaagttt ataaattcac cacgtatttccctcagctca catctttctg 300 agaacatgat aaattcatta cattgtgcta caccatatggggactttaga acatgttcgt 360 cttctttttt tttctttttt cttttttaca ttttaccttctcagtttaca aatacttgtt 420 agctttgcct ggatatgtaa cccaacacta tgatacaaattttgtcaatt ctctaaaatt 480 tttcaagttt gaaacaaatt aagatgtatg gttgtgggtgaattaatcta cattgatagg 540 aaatgcgtta aagtacatgc aaagcttatg aaattattgcaagtagtcat catgcctcag 600 tgagtcagtg tgcatacttg tagtgcataa ccaaattctttttcatatac tagaaagatt 660 caaagctgca aatgtgcatg tgaggttgat gaatggaatacacaataata catgacaata 720 aacacatatt aaagaattta gtgcaaaaaa attgtattgtcatggtacac attttaacaa 780 ttttctttac tttttataca ttgtaaatat taattaatatctaaaacaaa atataaagta 840 cgggaataaa atttaattgg ctaatgtgga aatggcatggagctaaatgt tctatatatg 900 gtcctaacgt ttaaagataa aatacatatg tgcttgtgttactaataata tctaaaagac 960 taagtagtat gtattaaata tgctagagaa cataagaaacttaaaaactt gacgtggcaa 1020 aatgggcgct taagcgacac atattgcact ttcagctgatcttattcccc cttttaaatt 1080 tcatgtcccc attttgatta tcacatggaa tcaaccacattatacctatg catccttgtg 1140 atttctgaaa ttaaactcga aagcaaccaa gtaaagaggagggggagaaa aagttagcag 1200 aaaacatgga tgatacatgt caataagctg ctagcataaccataagtgta gggcacgttt 1260 cttaggacaa tggtataaag ttacaaactg aaatatcattgttaggtcag tttgatataa 1320 tcagtttgga ttactataga cacttgtcat agtaaatagagatggatcat ttcttaagta 1380 gcatcactac tcttattcac gaatgtcttt gctaccctttctgttatact tttcctcttt 1440 ttctgtagaa agccatttgt ccttatatta tcattgtcaaattaaggatg cgtaatctac 1500 accctcactc aaaaactttg ataagataaa aacaaataaatccatgcctt tatagaacct 1560 tgtcaaaatt atgctacacc tgtgctagga acaccataccatcgtagctt acttgcacgc 1620 tttctgttag ccctttttcc taataaaaac gtattcgtccgtatcgttgt tatcgctttg 1680 atcgtgtggg tttcacttta tatccgttga aacctctgttaacacgtcca aattatttat 1740 atcggtatta tcactcaaga tcgtgcgggt tgttttgctttttacgtttc ttgaagcttc 1800 taaagagggg acaaacctat ataaatagga gaggagagcaccctctcaac tcagttcaaa 1860 attgaaaaaa aaaagaaaaa aaaagagaag aaaaaaaaacccatgggttg cgtagccgta 1920 gctttcccac catttccttc tctccgaagc ctcctcctctccgcttcctc ccgcgaaacc 1980 aaattccaaa gcatttgatc gaatttctcc caaacttttccagcgttttc aatttcgccc 2040 cgatttcggt tcgaaaaccc ctcgcgaatt catttcaaactcgtccgaga gcgcaatgga 2100 gcacgtgttc gccgtcgacg agatccccga cccgctgtgggctccgccgc cgccggtgca 2160 gccggcggcg gccgccggag tagatgacgt cggcgcggtgagcggcggcg ggttgctgga 2220 gcggtgcccg tcggggtgga acctcgagag gtttctggaggagctcgacg gcgtccctgc 2280 accggcggcg agcccggacg gcgcggcgat ttaccctagcccgatgccgg cggcggcggc 2340 ggaggcggcg gcgcgctgga gtaggggcta cggcgatcgtgaggcggtgg gggtgatgcc 2400 catgcccgcg gccgcgcttc cggcggcgcc ggcgagcgcggcgatggacc ccgtggagta 2460 caacgcgatg ctgaagcgga agctggacga ggacctcgccaccgtcgcca tgtggagggt 2520 actctctctc atctcgatcg ctgcttgctt tgcttgcttcatggcttgta cagttgtact 2580 ggtgggttca ccatttgggg tggtggtgat gggatggctgtggcgtaatt aagtgcaatt 2640 tttagggcat ttcctgtgat taactgtggc tagatggtcgcaatttagca tagatgtgac 2700 atatcctagc tgttactatg aatctggacc ggctctctgtccagattcat agtactagat 2760 gtgtcacatc cctctaaatc tcttatatta taaggagggagtataaatta attttataag 2820 agcacgcgtt gatgtcgata tccgcatcgt aagcccaggcactactcacg tgtgtgcttt 2880 cttatccata ctttaatatt gtcagagtgg gatgagacaaactttaatat tgtcggggtg 2940 tggtataata tttatattat ttccgtgtat agatttaggagtaatatgga ttaggattgc 3000 atggaggtgc agagacttta tgtgacttct tggagccgtgcattgcttga gtgcaaagtt 3060 aacaatttgg ttacatgttg caaaaatgat gtatagatcataggtcattg cacttatttt 3120 gggtggtcct aggcggtatg attcatggaa tattttttggaaattctgta ttttaccata 3180 tttgcattac ttttcttatt attgttgttt gaaggaattattaggtcaca tacccttgga 3240 agatgaaatt attttagtag aaaaaaagaa actgttatattggaatctgg taaatttgga 3300 cctagaaatt ctcaccagtc gattgtagat ggggaagcagagctttcttt ttagagattt 3360 gctccgctcc aacaaaaagt acctcgaggt actggtacctcatggtacca aatcgtttcc 3420 gatcgttgga tctaacaatg cacatcctgc ctaattagatccaacgatcg aaaatgattt 3480 ggtaccgtga ggtaccggta cctcgaggta ctttttgttggaccggagaa aatatcttct 3540 ttttatgtta gttttctaag tggggtatat aatttttgcaattggatatc atactttgaa 3600 ctcattatgt gggttcagtt tacaaatgac tacagaacatgttgatctga gcttttgcta 3660 gttgatttca gtttacaatg tgaaacggtt ccctatataagattataatg ccattagaac 3720 taattaacta tgagagtgtg tgtttagctc cgatagttattaagtccctt tgcattctga 3780 cttcaatttt tggcatgtcc atccatccac aggcctctggtgcaatacat tctgagagtc 3840 ctctaggcaa taaaacatca ctgagtatag ttggttccatcctgagttca cagaagtgca 3900 ttgaaggtat tctattatgc atatgtgctt agttaaatcttctcagtacc tatgagttat 3960 gacttatgag taatctctaa tgttgtaagc aaatctaatttttgcgtaat gtagttttca 4020 tattatatat atctgattgg attttccccc tatttcgacacattcaggta acgggatact 4080 agtgcagacc aagttaagtc ctggcccaaa tggaggatcaggcccatatg taaatcaaaa 4140 tacagatgct catgccaagc aagctacgag tggttcctcaagggagccat caccatcaga 4200 ggatgatgat atggaaggag atgcagaggc aatgggaaatatgatccttg atgaagaaga 4260 taaagtgaag aaaaggtaat atgtattctt ttgcttgtgtatttttattt ttcaattcaa 4320 cacatacaaa gagtaaacac tgagcattag cattagaaattaggggactt ttacatctat 4380 tgatttcctt ttttcttaga aatagctttt aagtaatatgctttagatta tcaagataat 4440 ggatccttag tttctttcta ggtgtttcat gtttgtactggatgtatttg attatataca 4500 acattctcac ttttttctta gaaatgcttg agctaatgcttgctaggtgt ttcaatgctt 4560 tatatacctg actgaatttt ggtaatgctt gttacaagctggtgcattaa ggataattat 4620 tgtttccgtg caagcagcta ttcatgcaaa aaaggaaaaatgcaacgtgt atgattagaa 4680 caatttagga ggcatttgct tcttgctttt cataacatgctgggaatatc atgtcctgtt 4740 gtgtctagtt gctttttcta catatgaaaa attgagtttatctactgtgg tctttttttc 4800 cgcagcagtc agacattcat gtcgcctttt tttgtgtaataaatacagcc ggatatttga 4860 gatttgagct tgtgttcttg tccaatttca ggaaggaatccaaccgggag tcagctagac 4920 gctcaagaag cagaaaggca gctcgcctaa aagacctggaggagcaggtt ttgtgtttta 4980 cactattcca tttgactgca caacaaagtt ttggaatatgtaagtaacaa gtgtaattgt 5040 tgctaaatca ttgcaggtat cactattaag ggttgaaaactcttctctgt tgaggcgtct 5100 tgctgatgca aatcagaagt acagtgctgc tgctattgacaatagggtac taatggcaga 5160 cattgaagcc ctaagagcaa aggtatgcaa ctgtttaagtgccttttagt cctctgtatg 5220 aactgaacct ctctttcaaa taggtatcca attatccatgtgcattgatt ctggtcagta 5280 ttgtgcatct ttcatggtgt agaaaaccgg aatattctacatatcaaaca tataccaaat 5340 tttcttggaa tgaaacgaac ttctagcatt tgttcttaaaatttggtaca ggagatattg 5400 caaatgttgt cctcttgctc cattcgaagg attaagttgtttgccatcta ttataacctg 5460 caacaattag actcacttgt tttgtcttga aacaaccgggtgtaactact tttctttttc 5520 ctgcaacgta ccaggtgtaa ataatcgctt gccgaatggtgataaccaat tcacacaatg 5580 gatcacaatc aattttaaca aagaacctga gctacactacactactgcgg tgtcgtatct 5640 tatagccata tgcttctaga ccacaactga aaattcatgaaccatgcgat gtgggttagc 5700 taacatcttg acatgattgc aggtgaggat ggcagaggagagtgtgaaga tggttacagg 5760 ggctagacaa cttcaccagg ccattcctga catgcaatctcccctcaatg tcaactctga 5820 tgcttctgtg ccgatccaga acaacaaccc aatgaactacttctccaacg ctaacaatgc 5880 cggtgttaac agcttcatgc accaggtttc tccagcgttccagattgtgg attctgtcga 5940 gaagattgac ccaacagatc cagtgcagct gcagcagcaacagatggcga gcttgcagca 6000 tcttcagaat agagcttgtg gtggcggcgc aagttcgaatgaatatacag catggggatc 6060 gtctctgatg gatgcaaatg agcttgtcaa catggagcttcagtagtagg agcatatcct 6120 aacaacatga tgagagcatt tggaggtgca aatttgcaacctgcaaatgc tgttttgtag 6180 tagtagttgt tgtcgctgtt tttgtctgaa actgtagtttctatggattt tggacttgct 6240 gaggaacatc tgcggctgtt gttgtttcaa attgagaaaatgagggacaa tgggacatgg 6300 tggtctccct taatatagcg aaaaatggtt ggaat 6335 41199 DNA Oryza sativa CDS (171)..(1004) 4 ggcacgaggc gatcaacacaaaaagcttct ctttcccttc tcctcctcgg tgatctgtct 60 cgccggggca tctcgaaaagcatccgactc cgacgccgcc gcgcgccacc acccggccga 120 tcgccgacgc cgcagccgctggaagcagca gggacgacgg agaatcggag atg gac 176 Met Asp 1 atc gag gcg ttcatc cac ggc gga agc ggg ggc ggc gac gcc gac gcc 224 Ile Glu Ala Phe IleHis Gly Gly Ser Gly Gly Gly Asp Ala Asp Ala 5 10 15 gac cac ccg ctc ggcatc ttc tcc gcc gcc gac ctc tcc ggc ttc ggc 272 Asp His Pro Leu Gly IlePhe Ser Ala Ala Asp Leu Ser Gly Phe Gly 20 25 30 ttc gcg gac tcg agc accatc aca ggg ggc att ccc aat cac ata tgg 320 Phe Ala Asp Ser Ser Thr IleThr Gly Gly Ile Pro Asn His Ile Trp 35 40 45 50 ccc cag tcc cag aac ctgaac gca cgg cat cct gcg gtc tcc acg aca 368 Pro Gln Ser Gln Asn Leu AsnAla Arg His Pro Ala Val Ser Thr Thr 55 60 65 att gag tcg cag tca tca atctgt gca gca gca agt ccc aca tca gct 416 Ile Glu Ser Gln Ser Ser Ile CysAla Ala Ala Ser Pro Thr Ser Ala 70 75 80 acc aat ctg aac atg aag gag agccaa act ctg gga ggc aca agt ggt 464 Thr Asn Leu Asn Met Lys Glu Ser GlnThr Leu Gly Gly Thr Ser Gly 85 90 95 tcg gat tct gaa agt gaa tcg ctg ttggat ata gag ggt ggt cca tgc 512 Ser Asp Ser Glu Ser Glu Ser Leu Leu AspIle Glu Gly Gly Pro Cys 100 105 110 gaa caa agc acg aac ccg ttg gac gtgaag aga gtg aga agg atg gtg 560 Glu Gln Ser Thr Asn Pro Leu Asp Val LysArg Val Arg Arg Met Val 115 120 125 130 tcc aat cgg gag tct gct cgg cgatcg agg aag aga aag caa gct cac 608 Ser Asn Arg Glu Ser Ala Arg Arg SerArg Lys Arg Lys Gln Ala His 135 140 145 tta gct gat ctc gag tca cag gttgac cag ctc cgg ggc gaa aac gca 656 Leu Ala Asp Leu Glu Ser Gln Val AspGln Leu Arg Gly Glu Asn Ala 150 155 160 tcg ctt ttc aag cag ttg acg gatgcc aac cag caa ttc aca act tct 704 Ser Leu Phe Lys Gln Leu Thr Asp AlaAsn Gln Gln Phe Thr Thr Ser 165 170 175 gtc acg gac aac aga atc ctc aaatca gac gtt gag gcc ctc cgg gtc 752 Val Thr Asp Asn Arg Ile Leu Lys SerAsp Val Glu Ala Leu Arg Val 180 185 190 aag gtg aag atg gcg gag gac atggtg gcg cgg ggg gcg ctg tcg tgc 800 Lys Val Lys Met Ala Glu Asp Met ValAla Arg Gly Ala Leu Ser Cys 195 200 205 210 ggg ctc ggc cac ctg ggc gggctg tcg ccg gcg ctg aac ccc cgg cag 848 Gly Leu Gly His Leu Gly Gly LeuSer Pro Ala Leu Asn Pro Arg Gln 215 220 225 gcg tgc cgc gtc ccc gac gtgctc gcc ggc ctg gac tac gcc ggc gac 896 Ala Cys Arg Val Pro Asp Val LeuAla Gly Leu Asp Tyr Ala Gly Asp 230 235 240 gac ccc ttc acg gcc ggg ctgtcc cag ccg gag cag ttg cag atg ccc 944 Asp Pro Phe Thr Ala Gly Leu SerGln Pro Glu Gln Leu Gln Met Pro 245 250 255 ggc ggc gag gtg gtt gac gcctgg ggc tgg gac aac cac ccc aac ggc 992 Gly Gly Glu Val Val Asp Ala TrpGly Trp Asp Asn His Pro Asn Gly 260 265 270 ggc atg tcc aag tgaaactactggtcctactt ctatgtcagc tcagctacgt 1044 Gly Met Ser Lys 275 ttgaaacgtgatgtgtccaa gtgaacggac ttgagttttt cagagtcctc gtgtcgaagt 1104 gtcatgcactcttccctatt cctgtaatag aactgactag ctaagagact gaaagtctga 1164 aactacgaagtataaatgtg gtggaatttg gaact 1199 5 278 PRT Oryza sativa 5 Met Asp IleGlu Ala Phe Ile His Gly Gly Ser Gly Gly Gly Asp Ala 1 5 10 15 Asp AlaAsp His Pro Leu Gly Ile Phe Ser Ala Ala Asp Leu Ser Gly 20 25 30 Phe GlyPhe Ala Asp Ser Ser Thr Ile Thr Gly Gly Ile Pro Asn His 35 40 45 Ile TrpPro Gln Ser Gln Asn Leu Asn Ala Arg His Pro Ala Val Ser 50 55 60 Thr ThrIle Glu Ser Gln Ser Ser Ile Cys Ala Ala Ala Ser Pro Thr 65 70 75 80 SerAla Thr Asn Leu Asn Met Lys Glu Ser Gln Thr Leu Gly Gly Thr 85 90 95 SerGly Ser Asp Ser Glu Ser Glu Ser Leu Leu Asp Ile Glu Gly Gly 100 105 110Pro Cys Glu Gln Ser Thr Asn Pro Leu Asp Val Lys Arg Val Arg Arg 115 120125 Met Val Ser Asn Arg Glu Ser Ala Arg Arg Ser Arg Lys Arg Lys Gln 130135 140 Ala His Leu Ala Asp Leu Glu Ser Gln Val Asp Gln Leu Arg Gly Glu145 150 155 160 Asn Ala Ser Leu Phe Lys Gln Leu Thr Asp Ala Asn Gln GlnPhe Thr 165 170 175 Thr Ser Val Thr Asp Asn Arg Ile Leu Lys Ser Asp ValGlu Ala Leu 180 185 190 Arg Val Lys Val Lys Met Ala Glu Asp Met Val AlaArg Gly Ala Leu 195 200 205 Ser Cys Gly Leu Gly His Leu Gly Gly Leu SerPro Ala Leu Asn Pro 210 215 220 Arg Gln Ala Cys Arg Val Pro Asp Val LeuAla Gly Leu Asp Tyr Ala 225 230 235 240 Gly Asp Asp Pro Phe Thr Ala GlyLeu Ser Gln Pro Glu Gln Leu Gln 245 250 255 Met Pro Gly Gly Glu Val ValAsp Ala Trp Gly Trp Asp Asn His Pro 260 265 270 Asn Gly Gly Met Ser Lys275 6 1362 DNA Oryza sativa CDS (23)..(907) 6 ggcacgaggt cggaggaagg cgatg atg aag aag tgc ccg tcg gag ctg cag 52 Met Met Lys Lys Cys Pro SerGlu Leu Gln 1 5 10 ctg gag gcg ttc atc cgg gag gag gcc ggc gcc ggc gaccgc aag ccc 100 Leu Glu Ala Phe Ile Arg Glu Glu Ala Gly Ala Gly Asp ArgLys Pro 15 20 25 ggc gtg tta tct ccc ggc gac ggc gcg cgt aag tcc ggc ctgttc tct 148 Gly Val Leu Ser Pro Gly Asp Gly Ala Arg Lys Ser Gly Leu PheSer 30 35 40 ccc ggc gac ggc gag atg tcc gtg ttg gat cag agt aca ctg gacgga 196 Pro Gly Asp Gly Glu Met Ser Val Leu Asp Gln Ser Thr Leu Asp Gly45 50 55 agc ggc ggc ggc cac cag ctg tgg tgg ccg gag agc gtc cgt acg ccg244 Ser Gly Gly Gly His Gln Leu Trp Trp Pro Glu Ser Val Arg Thr Pro 6065 70 ccg cgc gcc gcc gcc gcc ttc tcg gcc acg gcc gac gag cgg acg ccg292 Pro Arg Ala Ala Ala Ala Phe Ser Ala Thr Ala Asp Glu Arg Thr Pro 7580 85 90 gcg tcc atc tcc gat gac ccc aaa cca acc acc tca gcg aac cac gcg340 Ala Ser Ile Ser Asp Asp Pro Lys Pro Thr Thr Ser Ala Asn His Ala 95100 105 cct gaa agc gac tcg gac tcc gat tgc gat tcg ctg tta gaa gca gag388 Pro Glu Ser Asp Ser Asp Ser Asp Cys Asp Ser Leu Leu Glu Ala Glu 110115 120 agg agt cca cgc ctg cgt ggc acg aaa tcc aca gaa aca aag cga ata436 Arg Ser Pro Arg Leu Arg Gly Thr Lys Ser Thr Glu Thr Lys Arg Ile 125130 135 aga agg atg gtg tcc aac agg gag tcc gct cga cga tcc agg agg aga484 Arg Arg Met Val Ser Asn Arg Glu Ser Ala Arg Arg Ser Arg Arg Arg 140145 150 aag cag gca cag tta tct gaa ctc gaa tca cag gtc gag caa ctc aaa532 Lys Gln Ala Gln Leu Ser Glu Leu Glu Ser Gln Val Glu Gln Leu Lys 155160 165 170 ggc gaa aac tca tcc ctc ttc aag cag ctc aca gag tcc agc cagcag 580 Gly Glu Asn Ser Ser Leu Phe Lys Gln Leu Thr Glu Ser Ser Gln Gln175 180 185 ttc aat aca gcg gtc acg gac aac agg atc ctc aaa tcg gat gtagag 628 Phe Asn Thr Ala Val Thr Asp Asn Arg Ile Leu Lys Ser Asp Val Glu190 195 200 gcc tta aga gtc aag gtc aag atg gct gaa gac atg gtc gcg agggcc 676 Ala Leu Arg Val Lys Val Lys Met Ala Glu Asp Met Val Ala Arg Ala205 210 215 gcg atg tcg tgt ggc ctg ggc cag ctc ggg ctg gcg cca ttg ctcagc 724 Ala Met Ser Cys Gly Leu Gly Gln Leu Gly Leu Ala Pro Leu Leu Ser220 225 230 tcc agg aag atg tgc caa gct ttg gat atg ctc agt tta cca cggaac 772 Ser Arg Lys Met Cys Gln Ala Leu Asp Met Leu Ser Leu Pro Arg Asn235 240 245 250 gat gcc tgt ggt ttc aaa ggc ttg aac ctg ggt cga cag gttcag aac 820 Asp Ala Cys Gly Phe Lys Gly Leu Asn Leu Gly Arg Gln Val GlnAsn 255 260 265 tca ccg gtt caa agc gct gca agc cta gag agc ctg gac aaccgg ata 868 Ser Pro Val Gln Ser Ala Ala Ser Leu Glu Ser Leu Asp Asn ArgIle 270 275 280 tcc agc gag gtg acc agc tgc tcg gct gat gtg tgg ccttaagacactt 917 Ser Ser Glu Val Thr Ser Cys Ser Ala Asp Val Trp Pro 285290 295 catccgtgtt cgagagagct tgagattcta agaagcagcc ggtgagaatctgaaaaggct 977 agttgttcag tttcctattt ttagtttatg tttgaattct ctggctactaatgctcaaaa 1037 tctgggagag aatctaaatc gtttgggaca gataaaaaat tatgcgagaaggtgtagctg 1097 acagaaacct tcccaaacaa atctccatca gaacctatat gtaaagtaatacggtatcct 1157 ctgttactag gtgcatgtgc ataactgaca agctgctaag tactaggtactacagtctga 1217 ggcaagtatt tctggtgttt tggtgctgaa gaactatgtt ttagtgcgtttgatctgcgg 1277 caatcaaggc catctgatcg aaatttgatt ggtataaatc tgatcgaaatttgattggta 1337 taagtataat agtttgattt tgatc 1362 7 295 PRT Oryza sativa7 Met Met Lys Lys Cys Pro Ser Glu Leu Gln Leu Glu Ala Phe Ile Arg 1 5 1015 Glu Glu Ala Gly Ala Gly Asp Arg Lys Pro Gly Val Leu Ser Pro Gly 20 2530 Asp Gly Ala Arg Lys Ser Gly Leu Phe Ser Pro Gly Asp Gly Glu Met 35 4045 Ser Val Leu Asp Gln Ser Thr Leu Asp Gly Ser Gly Gly Gly His Gln 50 5560 Leu Trp Trp Pro Glu Ser Val Arg Thr Pro Pro Arg Ala Ala Ala Ala 65 7075 80 Phe Ser Ala Thr Ala Asp Glu Arg Thr Pro Ala Ser Ile Ser Asp Asp 8590 95 Pro Lys Pro Thr Thr Ser Ala Asn His Ala Pro Glu Ser Asp Ser Asp100 105 110 Ser Asp Cys Asp Ser Leu Leu Glu Ala Glu Arg Ser Pro Arg LeuArg 115 120 125 Gly Thr Lys Ser Thr Glu Thr Lys Arg Ile Arg Arg Met ValSer Asn 130 135 140 Arg Glu Ser Ala Arg Arg Ser Arg Arg Arg Lys Gln AlaGln Leu Ser 145 150 155 160 Glu Leu Glu Ser Gln Val Glu Gln Leu Lys GlyGlu Asn Ser Ser Leu 165 170 175 Phe Lys Gln Leu Thr Glu Ser Ser Gln GlnPhe Asn Thr Ala Val Thr 180 185 190 Asp Asn Arg Ile Leu Lys Ser Asp ValGlu Ala Leu Arg Val Lys Val 195 200 205 Lys Met Ala Glu Asp Met Val AlaArg Ala Ala Met Ser Cys Gly Leu 210 215 220 Gly Gln Leu Gly Leu Ala ProLeu Leu Ser Ser Arg Lys Met Cys Gln 225 230 235 240 Ala Leu Asp Met LeuSer Leu Pro Arg Asn Asp Ala Cys Gly Phe Lys 245 250 255 Gly Leu Asn LeuGly Arg Gln Val Gln Asn Ser Pro Val Gln Ser Ala 260 265 270 Ala Ser LeuGlu Ser Leu Asp Asn Arg Ile Ser Ser Glu Val Thr Ser 275 280 285 Cys SerAla Asp Val Trp Pro 290 295 8 12 DNA Oryza sativa 8 gctgagtcat ga 12 912 DNA Oryza sativa 9 catgagtcac tt 12 10 12 DNA Oryza sativa 10agtgagtcac tt 12 11 12 DNA Oryza sativa 11 ggtgagtcat at 12 12 12 DNAOryza sativa 12 ggtgagtcat gt 12 13 12 DNA Oryza sativa 13 gatgagtcat gc12 14 12 DNA Oryza sativa 14 aatgagtcat ca 12 15 12 DNA Oryza sativa 15agccacgtca ca 12 16 17 DNA Artificial Sequence Description of ArtificialSequence Artificially Synthesized Primer Sequence 16 tccaaymgng arwcngc17 17 21 DNA Artificial Sequence Description of Artificial SequenceArtificially Synthesized Primer Sequence 17 gtcctcygcc atcttcacct t 2118 21 DNA Artificial Sequence Description of Artificial SequenceArtificially Synthesized Primer Sequence 18 atgggttgcg tagccgtagc t 2119 21 DNA Artificial Sequence Description of Artificial SequenceArtificially Synthesized Primer Sequence 19 ttgcttggca tgagcatctg t 2120 17 DNA Artificial Sequence Description of Artificial SequenceArtificially Synthesized Primer Sequence 20 gaggatcagg cccatat 17 21 21DNA Artificial Sequence Description of Artificial Sequence ArtificiallySynthesized Primer Sequence 21 tcgctatatt aagggagacc a 21 22 24 DNAArtificial Sequence Description of Artificial Sequence ArtificiallySynthesized Primer Sequence 22 tgctccattg cgctctcgga cgag 24 23 23 DNAArtificial Sequence Description of Artificial Sequence ArtificiallySynthesized Primer Sequence 23 atgaattcgc gaggggtttt cga 23 24 25 DNAArtificial Sequence Description of Artificial Sequence ArtificiallySynthesized Primer Sequence 24 gtttgggaga aattcgatca aatgc 25 25 30 DNAArtificial Sequence Description of Artificial Sequence ArtificiallySynthesized Primer Sequence 25 atggtatggt gttcctagca caggtgtagc 30 26 18DNA Artificial Sequence Description of Artificial Sequence ArtificiallySynthesized Primer Sequence 26 aaaactgcag ttttctga 18 27 25 DNAArtificial Sequence Description of Artificial Sequence ArtificiallySynthesized Primer Sequence 27 aatggatccg cgaggggttt tcgaa 25 28 12 DNAOryza sativa 28 gcttcctcat ga 12 29 26 DNA Artificial SequenceDescription of Artificial Sequence Artificially Synthesized PrimerSequence 29 aaccatggtg ctggagcggt gcccgt 26 30 25 DNA ArtificialSequence Description of Artificial Sequence Artificially SynthesizedPrimer Sequence 30 aaccatggcg gcggaggcgg cggcg 25 31 23 DNA ArtificialSequence Description of Artificial Sequence Artificially SynthesizedPrimer Sequence 31 ccccatggag tacaacgcga tgc 23 32 25 DNA ArtificialSequence Description of Artificial Sequence Artificially SynthesizedPrimer Sequence 32 aaccatggtt ggttccatcc tgagt 25 33 25 DNA ArtificialSequence Description of Artificial Sequence Artificially SynthesizedPrimer Sequence 33 aaccatggct catgccaagc aagct 25 34 28 DNA ArtificialSequence Description of Artificial Sequence Artificially SynthesizedPrimer Sequence 34 aaccatggat gaagaagata aagtgaag 28 35 26 DNAArtificial Sequence Description of Artificial Sequence ArtificiallySynthesized Primer Sequence 35 taggatccgc tcctactact gaagct 26 36 27 DNAArtificial Sequence Description of Artificial Sequence ArtificiallySynthesized Primer Sequence 36 aaggatccaa tggagcacgt gttcgcc 27 37 26DNA Artificial Sequence Description of Artificial Sequence ArtificiallySynthesized Primer Sequence 37 aaggatccgg cggcggaggc ggcgcg 26 38 27 DNAArtificial Sequence Description of Artificial Sequence ArtificiallySynthesized Primer Sequence 38 gccggatcca gttggttcca tcctgag 27 39 26DNA Artificial Sequence Description of Artificial Sequence ArtificiallySynthesized Primer Sequence 39 aaggatcctg atgaagaaga taaagt 26 40 27 DNAArtificial Sequence Description of Artificial Sequence ArtificiallySynthesized Primer Sequence 40 aaggatccag gagtagatga cgtcggc 27 41 29DNA Artificial Sequence Description of Artificial Sequence ArtificiallySynthesized Primer Sequence 41 aaggatccag acgagatccc cgacccgct 29 42 28DNA Artificial Sequence Description of Artificial Sequence ArtificiallySynthesized Primer Sequence 42 tagagctcta cgccgccggc atcgggct 28 43 28DNA Artificial Sequence Description of Artificial Sequence ArtificiallySynthesized Primer Sequence 43 tagagctcta aaggatcata tttcccat 28 44 28DNA Artificial Sequence Description of Artificial Sequence ArtificiallySynthesized Primer Sequence 44 tagagctcta ggcggccgcc gccggctg 28 45 28DNA Artificial Sequence Description of Artificial Sequence ArtificiallySynthesized Primer Sequence 45 tagagctcta cggcggcggc ggagccca 28 46 25DNA Artificial Sequence Description of Artificial Sequence ArtificiallySynthesized Primer Sequence 46 aaaccatgga gcacgtgttc gccgt 25 47 26 DNAArtificial Sequence Description of Artificial Sequence ArtificiallySynthesized Primer Sequence 47 taggatccgc tcctactact gaagct 26 48 26 DNAArtificial Sequence Description of Artificial Sequence ArtificiallySynthesized Primer Sequence 48 aaaccatgga gggagaagct gagacc 26 49 30 DNAArtificial Sequence Description of Artificial Sequence ArtificiallySynthesized Primer Sequence 49 aaaggatcct acatatcaga agcggcggga 30 50 26DNA Artificial Sequence Description of Artificial Sequence ArtificiallySynthesized Primer Sequence 50 aaaccatgga tatagagggc ggtcca 26 51 30 DNAArtificial Sequence Description of Artificial Sequence ArtificiallySynthesized Primer Sequence 51 aaaggatcct acagcccgcc caggtggccg 30 52 21DNA Oryza sativa 52 gtttgtcatg gctgagtcat g 21

1. A DNA selected from the group consisting of: (a) a DNA encoding aprotein comprising the amino acid sequence set forth in any one of SEQID NOs: 2, 5, and 7; (b) a DNA comprising a coding region of thenucleotide sequence set forth in any one of SEQ ID NOs: 1, 3, 4, and 6;(c) a DNA comprising the amino acid sequence set forth in any one of SEQID NOs: 2, 5, and 7, in which one or more amino acids are substituted,deleted, added, and/or inserted, and encoding a protein that isfunctionally equivalent to a protein comprising the amino acid sequenceset forth in any one of SEQ ID NOs: 2, 5, and 7; and (d) a DNAhybridizing under stringent conditions with a DNA comprising thenucleotide sequence set forth in any one of SEQ ID NOs: 1, 3, 4, and 6,and encoding a protein functionally equivalent to a protein comprisingthe amino acid sequence set forth in any one of SEQ ID NOs: 2, 5, and 7.2. The DNA according to claim 1, which encodes a protein that binds tothe GCN4 motif or activates expression of rice seed storage protein. 3.The DNA according to claim 1 or 2, which is derived from rice plant. 4.A DNA encoding antisense RNA complementary to a transcription product ofthe DNA according to any one of claims 1 through
 3. 5. A DNA encoding anRNA having ribozyme activity that specifically cleaves a transcriptionproduct of the DNA according to any one of claims 1 through
 3. 6. A DNAencoding an RNA that suppresses the expression of the DNA according toany one of claims 1 through 3 in plant cells by co-inhibition effects,and having 90% or more homology with the DNA according to any one ofclaims 1 through
 3. 7. A DNA encoding a protein having a dominantnegative phenotype of a protein encoded by the DNA according to any oneof claims 1 through 3 which is endogenous in plant cells.
 8. A vectorcontaining the DNA according to any one of claims 1 through
 3. 9. Atransformed cell retaining the DNA according to any one of claims 1through 3 or the vector according to claim
 8. 10. A protein that isencoded by the DNA according to any one of claims 1 through
 3. 11. Amethod of producing the protein according to claim 10, the methodcomprising steps of culturing the transformed cell according to claim 9and collecting the expressed protein from said transformed cell or theirculture supernatant.
 12. A vector containing the DNA according to anyone of claims 4 through
 7. 13. A transformed plant cell retaining theDNA according to any one of claims 1 through 7 or the vector accordingto claim 8 or
 12. 14. A transformed plant containing the transformedplant cell according to claim
 13. 15. A transformed plant that is aprogeny or clone of the transformed plant according to claim
 14. 16. Areproductive material of the transformed plant according to claim 14 or15.
 17. An antibody that binds to the protein according to claim
 10. 18.A plant having on its genome a DNA construct in which the DNA accordingto claim 1 is operably connected downstream of an expression controlregion and a DNA construct in which a foreign gene is operably connecteddownstream of an expression control region having the target sequence ofthe protein according to claim
 10. 19. The plant according to claim 18,wherein the target sequence is a sequence containing the GCN4 motif. 20.The plant according to claim 19, wherein the GCN4 motif has the sequenceset forth in any one of SEQ ID NOs: 8, 13, and
 14. 21. The plantaccording to claim 18, wherein the target sequence is a sequencecontaining a G/C box.
 22. A method of producing the plant according toany one of claims 18 through 21, the method comprising a step ofcrossing a plant having on its genome a DNA construct in which the DNAaccording to claim 1 is operably connected downstream of an expressioncontrol region, with a plant having on its genome a DNA construct inwhich a foreign gene is operably connected downstream of an expressioncontrol region containing the target sequence of the protein accordingto claim 10.